Haider al-Abadi said he would "never discuss" the result and called on the Kurds to initiate dialogue "in the framework of the constitution".
He also vowed to "impose Iraq's rule" in all parts of the Kurdistan Region.
The referendum's result has yet to be announced, but people were expected to overwhelmingly back independence.
Kurdish leaders have said a "yes" vote will give them a mandate to start negotiations on secession with the central government in Baghdad and with neighbouring countries.
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