After days of crisis talks, the network confirmed on Friday morning that Stefanovic had left its flagship breakfast program, Today, as host effective immediately.
In a video on The Karl Stefanovic Show YouTube page shortly after the announcement, Stefanovic said he was now "truly independent".
"So I'm free. Truly independent," he said.
He thanked his free-to-air audience of 21 years for "riding the ups and downs".
"It wasn't easy these last couple of days, but we roll on."
It follows the release of his now-deleted podcast episode with British far-right figure Tommy Robinson, who has been accused of fuelling violent anti-immigration protests in the UK.
In his statement, Stefanovic said he believes in "different perspectives".
"So here's what I believe in: the public deserve to hear perspectives. … so on my show I've spoken to people who have different perspectives, I know that winds some people up," he said.
"Sometimes I agree with everything a guest says, sometimes I don't. But importantly you get to make up your mind."
"Freedom of speech, here and around the world, is what this show is about. You don't have to listen to my show, you have the power."
Earlier on Friday, a Nine spokesperson said: "Nine Entertainment and Karl Stefanovic have agreed that it is no longer possible for him to continue hosting Today at the same time as his independent podcast.
"While Karl and Nine had previously agreed he would leave Today at the end of this year, they have subsequently decided he will leave the Network immediately."
Stefanovic's Today co-host Sarah Abo delivered a tearful message to viewers on the show on Friday morning.
"Karl has, of course, woken up with today for 21 years so we know this is a huge change for our viewers and of course, for us, not just here on the desk, but behind the scenes," she said.
"He's our mate and he's our colleague, and it goes without saying that Karl's presence will be missed."
Abo said those who have worked alongside Stefanovic know how hard he works.
"We all wish it wasn't ending like this and that we could say goodbye to you Karl, our friend, in a different way, but ... we wish you all the best," she said.
Prime Minister Anthony Albanese said it was unfortunate how Stefanovic's TV career had ended.
"Words do matter, and what people who are very prominent in our public discourse do, and how they conduct themselves matters as well," he told ABC News Breakfast.
In a note to staff, Nine's news and current affairs executive director, Fiona Dear, said protecting freedom of speech was core to the network.
"We have a rich history of interviewing controversial figures, and journalists have an obligation to ask difficult questions," she said in the internal note.
"We welcome open debate and challenging norms with constructive, balanced arguments.
"As journalists, we play a critical role in society to question, challenge, and hold people to account regardless of where they come from or which political party or views they represent."
She also acknowledged the challenging period for the Today show team and said the network will share plans for the show "very soon".
The episode with Robinson, in which Stefanovic said he admired the controversial figure's "tenacity" and "courage", was published on Tuesday evening but was pulled down by Wednesday morning.
However, the episode was resurrected on YouTube by Pauline Hanson, who Robinson praises during the interview.
The decision to ditch Stefanovic was not entirely surprising, with the host moving away from his playful breakfast larrikin persona to embrace a more controversial role as a podcaster, the University of Sydney's Catharine Lumby told AAP.
"We're living in a very unhinged world, where polarisation is rife, driven by social media and their algorithms and that includes podcasting more broadly," Professor Lumby said.
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