Villa lost 2-0 to fellow strugglers Hull City on Tuesday, their 10th league game without a win.
The 45-year-old Scot, who was appointed in 2012, signed a contract extension through until June 2018 on 17 September.
First-team coach Scott Marshall and goalkeeping coach Andy Marshall have taken caretaker charge.
Fans called for Lambert’s sacking during the game against Hull. Local newspaper, the Birmingham Mail, tweeted a picture of its front page saying that ‘Lambert Must Go’ with the accompanying hashtag, #lambertout.
Following the Hull game, Lambert said: “I was aware of [the discontent] months ago. I’ve told the players they’ve got 13 cup finals.”
Chief executive Tom Fox had defended Lambert and vowed to keep him as manager.
He told BBC sport: “When things aren’t going well, fans bay for that type of blood,” Fox said. “That’s not the way that I or the owner are going to make a decision. It’s a false narrative.”
But on Wednesday, Villa released a statement confirming the manager’s exit.
“Aston Villa Football Club has parted company this evening with manager Paul Lambert,” it read.