The ministry claimed the image showed an IS convoy leaving a Syrian town last week aided by United States forces.
But the Conflict Intelligence Team, described by The Guardian as "Russian online investigators who fact-check claims by the Russian military", refuted the Ministry's claims in a Facebook message of its own.
The ministry said an employee had mistakenly attached the photo.
The original game footage, which was cropped in the ministry's post, included a red "FIRE" button in the bottom-right corner as well as a disclaimer in the top-right corner that read: "Development footage".
Russian Federation re-published the video writing, "The U.S. are actually covering the ISIS combat units to recover their combat capabilities, redeploy, and use them to promote the American interests in the Middle East". "All content subject to change".
"However, the USA command's refusal to carry out strikes on the convoys of ISIL terrorists retreating from Albu Kamal on November 9 is an objective fact reflected in the transcripts of the talks and therefore, fully known to the US side", Interfax news agency quoted the ministry as saying on Tuesday. The statement wasn't even fully cropped out of the images that Russian Federation tweeted.
"Such actions by the coalition show that the rapid advance of the Syrian troops in Abu Kamal have disrupted US plans to create pro-American authorities beyond the control of the Syrian government, and to manage the territories on the eastern bank of the Euphrates", it said. "As proof, the MoD's social media account posted what they claimed were Russian drone photos of the convoy heading towards the Syrian-Iraqi border", the group said.
"The defense ministry is looking into a civilian employee of one of the departments who attached photos to the defense ministry's statement by mistake", a new statement was quoted by Russian agencies as saying.
"I certainly can't verify, but I've seen the report that one of the pictures came from a video game".