"In the issue of the nuclear deal, the current administration has committed several violations, the latest of which is the renewal of the 10-year sanctions," Khamenei said in a televised speech to thousands of members of the Basij Islamist volunteer militia.
"If these sanctions happen, it is absolutely a breach of the JCPOA," he added, referring to last year's deal with world powers under which sanctions were eased in exchange for curbs to Iran's nuclear programme.
"They must know that the Islamic republic will react against it."
The US Congress last week voted to renew long-standing sanctions linked to Iran's ballistic missile tests and human rights record that pre-date the controversy around Iran's nuclear ambitions.
The 10-year sanctions must still be approved by the Senate and President Barack Obama by the end of the year.
Washington says these sanctions have nothing to do with the nuclear deal, but Iran says the continuing restrictions go against the spirit of the agreement, particularly since they have discouraged international banks from returning.
"The JCPOA must not become a tool for pressuring the Iranian people," Khamenei said.