The decision, which contradicted the conciliatory tone set by Mr Putin just a day earlier, caused consternation in the West, which fears the referendum will tear Ukraine apart.
US Deputy Secretary of State William Burns said Russia was heading down a "dangerous and irresponsible path" and the situation in Ukraine was "extremely combustible".
Denis Pushilin, a leader of the self-declared separatist Donetsk People's Republic, said the "People's Council" had voted unanimously to hold the plebiscite as planned.
He added: "Civil war has already begun. The referendum can put a stop to it and start a political process."
The announcement coincided with a sharp change of tone from Moscow, which had signalled a pullback from confrontation on Wednesday with Mr Putin's call for the vote to be delayed and a declaration that troops were withdrawing from Ukraine's border.
In Kiev, officials promised to press on with their "anti-terrorist campaign" to retake control of the eastern regions of Donetsk and Luhansk regardless of the rebels' decision on the vote.
Political analysts said Mr Putin may have expected the rebels to go ahead with the referendum, showing that they were not under his orders. By distancing himself from a process that will not be recognised by the West, Mr Putin may also be hoping to avoid further sanctions as they begin to affect the Russian economy.
Mr Putin's spokesman said the Kremlin needed more information about the rebels' decision. He said the rebel statement came only after Kiev had declared it would press on with its military operation, implying Ukraine was to blame for the rebels' refusal to heed Mr Putin.
Nato and the US have both said they have seen no sign of a Russian withdrawal from the frontier despite Mr Putin's announcement he had pulled back troops.
Nato has accused Moscow of using special forces in the separatist takeover of mainly Russian-speaking eastern Ukraine after annexing Crimea from Ukraine in March. Mr Putin acknowledged his troops were active in Crimea after initially denying any role there, but has said that they are not involved in eastern Ukraine.
The referendum has become seen as a vital step by many in Ukraine's industrial east, fired up over what the rebels, and Moscow, call the "fascist" government in Kiev that took over after street protests ousted a pro-Moscow president in February.
While many Russian speakers in Ukraine fear discrimination under the new leadership, quite how many support the separatists, many of whom say their ultimate aim is to join Russia, is not so clear.
Mr Putin said his call for the postponement of the referendum would open the way to negotiations on cooling down a crisis that has led to dozens of deaths in clashes between troops and separatists in eastern Ukraine and rival groups in the southern port of Odessa.
Maria Lipman, an expert at the Carnegie Center think-tank in Moscow, said Mr Putin would have known his request for the referendum to be postponed would be rebuffed.
In a further shift away from reconciliation, Mr Putin oversaw test launches of military rockets during training exercises held across Russia yesterday.
Meanwhile, the Russian ambassador to Paris said Mr Putin, who had been shunned by Western leaders since the Crimean takeover, would join them at a ceremony next month to mark the 70th anniversary of the Normandy landings.