The 19-year-old 'Beauty And A Beat' hitmaker, who joined the micro-blogging site in 2009, has beaten other stars, including Lady Gaga and Taylor Swift, to the milestone.
"40 million strong... Beliebers thank you. I love you and I am grateful (sic)," Bieber posted on Twitter.
Bieber's Twitter record comes soon after 'White House Down' actor Channing Tatum revealed he was worried about the young singer.
The 33-year-old actor thinks the 'Baby' hitmaker, who was discovered at the age of 14, found fame too young and hopes he doesn't go off the rails like many child stars do.