The group's much-discussed first ad ran ahead of Gen. David Petraeus's congressional testimony last week and said: "General Petraeus or General Betray Us?"
The new ad takes aim at McConnell for leading Republican opposition to an amendment that failed to pass the Senate this week.
That amendment, sponsored by Sens. Jim Webb, Democrat of Virginia, and Chuck Hagel, Republican of Nebraska, would have mandated that troops spend an equal amount of time at home as they do in combat and would have sped up a redeployment of forces from Iraq.
Republicans and Pentagon officials said that it would harm the mission in Iraq and hamstring the military's rotation of troops.
The ad, focusing on McConnell, says: "Republicans voted against a plan that would give our soldiers reasonable time to spend with their families before having to return to war. This week, Republicans who brought us this awful war turned their backs on the men and women who fight it. Mitch McConnell. The Republicans. A betrayal of trust."
MoveOn is spending $100,000 on the ad buy, which is airing in Kentucky and on CNN nationally.
By Silla Brush