The video, entitled 'A Message in Blood', was posted online hours after another video purporting to show the mass execution of up to 250 Syrian soldiers in the desert.
It shows a group of captured men, believed to be Kurdish fighters, in orange jumpsuits sat in front of an Isis flag.
Kurdish 'peshmerga' fighters are trying to counter the militant's advances in northern Iraq. They are backed by US air strikes on Isis targets in the country.
Another man is then shown kneeling on the floor in front of a mosque with three militants behind him in Mosul, Iraq's second biggest city, which was overrun by Isis when their insurgency began.
The victim is then beheaded and the fighters warn others will face the same fate should Kurdish leaders choose to continue an alliance with the US.
It comes after the group posted a video on YouTube showing the American journalist James Foley being beheaded by a militant, who said his killing was in response to air strikes. The fighter, who is believed to be British, also threatened the life of another journalist, Steven Sotloff.
Video footage posted on Thursday appeared to show Syrian soldiers being marched through the desert in their underwear after Isis seized the Tabqa air base in Raqqa.
The Syrian Observatory for Human Rights said the soldiers were captured when they attempted to flee the fallen air base in Raqqa after days of fierce fighting. They put the death toll at 120.
On Thursday, President Barack Obama admitted the US does not yet have a strategy for confronting Isis militants in Syria.
He approved air strikes against Isis in Iraq earlier this month following the beheading of Mr Foley but has not made public any plans for across the border
He told a press briefing in Washington: 'I think what I've seen in some of the news reports suggests that folks are getting a little further ahead of where we're at than we currently are.
'Our core priority right now is just to make sure that our folks are safe and to do an effective assessment of Iraqi and Kurdish capabilities.'
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