"This is an illegitimate decision," Yatsenyuk told reporters after talks with EU leaders holding an emergency meeting on the Ukraine crisis.
"We urge the Russian government not to support those who advocate separatism," he added.
Pro-Moscow lawmakers in Crimea made the request to Russian President Vladimir Putin and said they would put the question to a referendum on March 16 as Russian forces maintained their grip on the strategic Black Sea peninsula.
The referendum, brought forward from a planned March 30 date, will also ask if residents want to a return to a previous 1992 constitution that gave the region sweeping autonomy.
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Kiev was ready to sign the pact which would have put Ukraine on course for closer ties with the EU.
"The Ukraine government is determined to sign the (EU) association agreement... We are ready as soon as possible," he said.
"We are ready for cooperation but we are not ready to surrender and be the subordinate of Russia," he said.
Putin has been building up "a new wall of intimidation, of military aggression," he said, calling on the Russian president instead to pull that wall down and to build a "new relationship".
Kiev said it had refused to be provoked by Russian actions and wanted above all to solve the issue peacefully.
"But in case of further escalation and military intervention... The Ukraine government and military will act in accordance with the constitution.
EU leaders meanwhile continued their talks amid differences over how far to go to sanction Moscow for its actions in Ukraine.
Moscow satellites, want a much tougher line with sanctions.