May, speaking to Parliament, said the response would include a halt to high-level meetings between British and Russian officials and the cancellation of a planned visit to Britain by Russian Foreign Minister Sergei Lavrov.
She also said the royal family and government ministers would boycott this summer's World Cup soccer tournament in Russia. More countermeasures - some clandestine - are under consideration.
The prime minister repeated the conclusion of British investigators that Russia had either deployed or lost control of a dangerous nerve agent used in the attack targeting former spy Sergei Skripal, 66, and his daughter, Yulia, 33.
"Instead, they have treated the use of a military-grade nerve agent in Europe with sarcasm, contempt and defiance," she told lawmakers.
The British leader gave no further details on the Russian diplomats ordered out of the country but said they were deemed "undeclared intelligence officers." She called it the largest expulsion of Russian diplomats from Britain since Cold War-era retributions in the 1980s.
Russian Foreign Ministry spokeswoman Maria Zakharova said the list included officials from the London embassy's military attache's office.
The Russians denounced May's accusation as an "unprecedentedly crude provocation" and a new blow to relations.
"The British government has opted for confrontation with Russia," a Foreign Ministry statement said. "To be sure, our response will not be long in coming."
Skripal, a former Russian double agent, was jailed in Russia in 2006 for selling state secrets to British intelligence for 10 years, but he was released in 2010 as part of a high-profile spy swap.
He and his daughter were found comatose on a park bench in the quiet town of Salisbury, near Stonehenge, on March 4. They remain in critical condition. Detective Sgt. Nick Bailey of the Wiltshire Police, who went to the aid of Skripal and his daughter, was hospitalized but is in stable condition. An additional 18 people were treated and released, police said.
"It is not in our national interest to break off all dialogue between the United Kingdom and the Russian Federation," May said in Parliament. "But in the aftermath of this appalling act against our country, this relationship cannot be the same."
The prime minister framed the use of a nerve agent in Salisbury as the first chemical weapons attack on NATO territory since the organization's founding.
She said Britain sought support from NATO, the United Nations, the European Union and the United States, but she did not mention any specific requests for allies to join in the reprisals.
In Washington, the White House said in a statement that the United States shares Britain's assessment that Russia was responsible for the attack and supports Britain's decision to expel Russian diplomats.
The attack "fits into a pattern of behavior in which Russia disregards the international rules-based order, undermines the sovereignty and security of countries worldwide, and attempts to subvert and discredit Western democratic institutions and processes," White House press secretary Sarah Huckabee Sanders said.
Speaking at the U.N. Security Council on Wednesday, U.S. Ambassador Nikki Haley echoed Britain's conclusion that Russia was responsible for the attack.
The two statements were stronger than President Trump's remark Tuesday, when he said that "as soon as we get the facts straight, if we agree with them, we will condemn Russia or whoever it may be."
As May spoke, a British counterterrorism unit opened a second investigation into the unexplained death of an enemy of Russian President Vladimir Putin. The Telegraph newspaper described Nikolai Glushkov, 68, as "the right-hand man of the deceased oligarch Boris Berezovsky, Mr Putin's one-time fiercest rival." Glushkov was found dead at his London home Monday.
In Parliament on Wednesday, lawmakers took turns condemning the nerve-agent attack and calling Russia a rogue state that should be punished. Several returned to May's promise that there is no place for corrupt Russians and their money in Britain, and they urged using existing laws to go after individuals with "unexplained wealth."
British politicians and commentators said May could further employ a range of diplomatic and financial sanctions - from clamping down on Russian oligarchs' property-buying binge in London to expelling more embassy personnel.
Options that could be taken in concert with allies may be more limited.
Britain's Foreign Office called Wednesday for an urgent meeting of the U.N. Security Council to update members on the nerve-agent investigation. Russia, as part of the permanent five nations on the council, holds veto power over any possible U.N. moves to come.
In Brussels, home of the NATO defense alliance, British diplomats made clear they believed the attack had security consequences for all 29 members.
But the British diplomats held back from triggering the formal processes that could escalate the NATO response and pull other nations into a confrontation with Russia, a NATO official said, speaking on the condition of anonymity to discuss a closed-door meeting.
The attack comes at a delicate moment for members of the Western security alliance that usually have little trouble presenting a united front against Russia. E.U. leaders plan to discuss the issue at a previously scheduled summit next week, but Britain's in-process divorce from the bloc limits the European appetite to make sacrifices for London. Europe is also fighting with Washington about Trump's tariffs on steel and aluminum, a dispute that could impede any transatlantic response to the poisoning.
"At a time of fake news spreading, meddling in our elections, and attacks on people on our soil with nerve agent, the response must not be transatlantic bickering but transatlantic unity," European Council President Donald Tusk wrote on Twitter. He offered May his "full solidarity."
But a European diplomat said it would be "ironic" for May to seek E.U. sanctions against Russia after Brexit voters rejected the support and unity of the E.U. The diplomat, who spoke on the condition of anonymity to discuss a delicate issue, said that although many European leaders are appalled by the nerve-agent attack, the unity required to apply sanctions probably would not be achievable.
The Russian response signaled a tit-for-tat measures to come. "The British, without presenting evidence of any kind, have said Russia is guilty. This is without precedent," said Valentina Matvienko, speaker of the upper house of the Russian parliament. "We, Russia, should therefore react swiftly, strongly, and in kind."
In a conference call with journalists in Moscow, Kremlin spokesman Dmitry Peskov said that theories surrounding Skripal's poisoning are not the Kremlin's concern. He reiterated that Moscow's official position asserting noninvolvement in the poisoning and demanding proof of Britain's accusations has been delivered through diplomatic channels.
Peskov said Moscow does not accept London's accusations and hopes the West will come to its senses and engage Russia in a joint investigation into the poisoning of Skripal.
(Except for the headline, this story has not been edited by NDTV staff and is published from a syndicated feed.)