The 'Never Say Never' singer reached the milestone in the aftermath of performing in Times Square on New Year's Eve (December 31).
Bieber thanked his fans for their support in his own tweet on New Year's Day (Sunday).
"NEVER SAY NEVER!! ... thank you and all 16 MILLION BELIEBERS! LOVE U," the popstar wrote.
Bieber's Twitter account has been flooded by well-wishes from his fans since he surpassed the 16m mark.
The 17-year-old appeared at Times Square on Saturday for Dick Clark's New Year's Rockin' Eve alongside stars such as Lady GaGa, Drake and Florence + the Machine.
Bieber also teamed with friend Jaden Smith to unveil the new track 'Happy New Year'.
> Justin Bieber covers The Beatles' 'Let It Be' in New York - video
> Justin Bieber's grandparents recovering after car accident
Watch Justin Bieber perform on Dick Clark's New Year's Rockin' Eve below: