Ambassador Kang Chol was declared "persona non grata" and asked to leave Malaysia within 48 hours.
Kim Jong Nam, the leader's half-brother, was murdered on Feb. 13 at Kuala Lumpur International Airport, after being assaulted by two women who Malaysian police believe smeared his face with VX, a nerve agent classified by the United Nations as a weapon of mass destruction.
The ambassador had said last month that Pyongyang "cannot trust" Malaysia's handling of the investigation, and accused the country of "colluding with outside forces", in a veiled reference to bitter rival South Korea.
Malaysian Prime Minister Najib Razak called the comments "diplomatically rude". Foreign Minister Anifah Haji Aman said in a statement on Saturday that Malaysia had demanded an apology from the ambassador, but none was forthcoming.
"Malaysia will react strongly against any insults made against it or any attempt to tarnish its reputation," Anifah said.
Earlier this week, Malaysia said it would cancel visa-free entry for North Koreans entering the country from March 6. Anifah said in the statement that this move was "an indication of the government's concern that Malaysia may have been used for illegal activities."
Malaysia on Saturday rejected any suggestion it may have violated sanctions imposed by the United Nations on North Korea, after a Reuters report said North Korea-linked firms were running an arms network in the country.
Reuters reported on Monday that North Korean intelligence agents used front companies to run an arms sales operation out of Malaysia under a brand called Glocom.
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