The singer, who had attracted 3.1 million followers, had spoken about leaving Twitter in April this year, saying during an interview that the site is "pretty much done".
Yesterday, Mayer shut his account after tweeting: "What does this button do?"
E! Online reports that Mayer's spokesman confirmed the deletion: "With the Battle Studies tour now at a close and a return to the studio planned, John has discontinued his Twitter account. However, he continues to communicate with his fans via his blog as he always has."
Mayer is reportedly maintaining a low-profile after attracting criticism for comments made in a Playboy interview.