The 'Beauty and a Beat' singer has amassed over four thousand more followers than the 'Edge of Glory' hitmaker, bringing his total to 33,331,355 and counting compared to Gaga's 33,327,342 on the social networking site.
Gaga had held the title for almost three years since overtaking Britney Spears in 2010.
The 'Born This Way' star was also the first Twitter user to cross the 10 million followers threshold, and broke her own record by becoming the first user to cross 20 million followers and then 30 million.
Gaga previously tweeted about Justin's growing Twitter following, saying: ''So proud of @justinbieber and all the Beliebers! I'm only happy to see your fans growing in size, you all deserve it! Monsters support you!''
Both singers have prominent fan bases on the website, with Justin's fans nicknamed 'Beliebers', and Gaga's branded 'Little Monsters'.
The 10 most followed Twitter accounts:
1. Justin Bieber (@justinbieber)
2. Lady Gaga (@ladygaga)
3. Katy Perry (@katyperry)
4. Rihanna (@rihanna)
5. Barack Obama (@BarackObama)
6. Britney Spears (@britneyspears)
7. Taylor Swift (@taylorswift13)
8. YouTube (@YouTube)
9. Shakira (@shakira)
10. Kim Kardashian (@KimKardashian)
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