Kutcher surpassed that benchmark in the early morning hours Friday, narrowly edging out the Time Warner Inc.-owned network.
The actor had long trailed CNN, but he staged a rally in recent days that captured the attention of the Web.
After his come-from-behind win, Kutcher celebrated by tweeting "Victory is ours!" and webcasting a video live on ustream.tv.
At the same time, Oprah Winfrey made her entry to Twitter, joining the site and holding a Twitter special on her show Friday with Kutcher as a guest. She gained 100,000 followers in less than a day.
Kutcher, CNN and Winfrey pledged to mark the occasion by purchasing mosquito bed nets to combat malaria. Kutcher donated $100,000 to the Malaria No More Fund, the charity said Friday.
Though celebrities have become commonplace on Twitter -- where people "follow" each other's updates of 140-characters or less -- Winfrey's entry was sure to cause ripples.
The talk show host was welcomed onto the site with messages from George Stephanopoulos, Ellen DeGeneres, Jimmy Fallon and Shaquille O'Neal.
What the El!?! Editorial:
Ashton Kutcher and Justin Timberlake are pretty cool dudes, don't you think?