Alexander Borodai, the former PM of the self-declared Donetsk People’s Republic, confirmed the news but denied reports Igor Strelkov had been wounded.
There has been heavy shelling both in Donetsk and Luhansk as Ukrainian forces battle the separatists.
The news came with a disputed Russian aid convoy stationed near the border.
Alexander Borodai confirmed Igor Strelkov’s departure to Russian media but gave no reason.
He said reports that Igor Strelkov, a Russian citizen, had been injured were “total rubbish”.
“You probably already know that he, like myself, has left his post,” Alexander Borodai said in a video posted by Russia’s pro-Kremlin Life News website.
“The [Donetsk People’s Republic] already has a new defense minister.”
Alexander Borodai said the new minister went by the nom de guerre Tsar. His name was given by other sources as Vladimir Kononov.
There have been two other high-profile resignations of rebel leaders in the past week.
Alexander Borodai handed over to Alexander Zakharchenko as “prime minister” in Donetsk and the rebel chief in Luhansk, Valery Bolotov, said he was temporarily handing over to his defense minister, Igor Plotnitskiy.
The Russian aid convoy of at least 260 trucks, which moved towards the border on Thursday, has now halted.
There is continuing confusion over the final destination.
Russia has dismissed as absurd claims that its convoy is a pretext to send military supplies to the rebels.
However, Ukraine has said the Russian convoy must be inspected by international monitors before it can be let in.
Andriy Lysenko, a spokesman for Ukraine’s National Security and Defense Council, said that if this did not happen, “movement of the convoy will be blocked with all the forces available”.
Artillery fire could be heard all around Donetsk on Thursday, with the authorities urging people to stay off the streets.
Two shopping centers were reportedly hit and at least one person killed.
The situation was reported to be desperate in Luhansk, where civilians have been short of water, food and electricity for more than a week. Phone lines are also down as shelling continues.
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