Ahead of the vote, the country's acting president, Oleksander Turchinov, warned secession from Ukraine would "mean the complete destruction of the economy, social programmes and life in general for the majority of the population in these regions".
The referendum, which is not recognised by Kiev or the West, seeks approval for declaring so-called sovereign people's republics in the troubled regions of Donetsk and Luhansk, in eastern Ukraine, where pro-Russia insurgents have seized government buildings and clashed with police and the army for weeks.
Separatists have so far refused to call off the referendum and ignored a request from Russian President Vladimir Putin to "postpone" the vote, putting the country on the brink of full-blown civil war.
Mr Turchinov's comments came as German Chancellor AngelaMerkel and French President Francois Holland called on the Kremlin to "send more signals of de-escalation" and reduce the presence of Russian troops on the Ukrainian border.
In a joint statement, Germany and France also warned that if 25 May Ukrainian elections failed to go ahead as planned, they would be "ready to take further sanctions against Russia" and urged Kiev to abstain from military action ahead of the vote, except "proportionate" operations to protect civilians or property.
"If there is not an internationally recognised presidential election, that would lead unavoidably to a further destabilisation of the country. Germany and France are in agreement that if that is the case, then corresponding consequences would be drawn as outlined by the European Council on March 6, 2014," the statement said.
Both leaders said they viewed tomorrow's referendum as "illegal" and insisted the most important goal at the moment is to ensure the presidential elections take place as planned.
Mr Hollande added: "This presidential election must take place in regular conditions so that the president then has legitimacy necessary for his work."
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Ukraine crisis: Voting for self-rule 'would be a disaster'