Ms Abedin's announcement comes after a report emerged that he had allegedly exchanged sexual photos with a woman.
He appeared to have sent a photo of himself in his underpants with his toddler son nearby.
The former congressman resigned in 2011 after sending explicit photos to women by text message.
"After long and painful consideration and work on my marriage, I have made the decision to separate from my husband," Ms Abedin said in a statement.
"Anthony and I remain devoted to doing what is best for our son, who is the light of our life. During this difficult time, I ask for respect for our privacy."
She is one of Mrs Clinton's most trusted advisers and Republican Donald Trump said it showed a lack of judgement on her part, with Mr Weiner possibly having access to classified information.
Analysis - Tara McKelvey, BBC News White House reporter
Why did it take so long? That's what everybody wants to know. Abedin is now officially separated from her husband, ending (at least temporarily) a marriage that has been scrutinised in public for years.
She appeared in a fascinating documentary, Weiner, in which she stood near her husband with arms crossed, looking really angry, for much of the time.
After an earlier sexting scandal surrounding Weiner, she said that her work as an adviser to Hillary Clinton became a refuge. "My compass was my job," she told the New York Times.
Clinton is now in the final stretch of her campaign. Abedin will have to focus - and try not to think about her personal problems. That may be a relief.
The New York Post reported that Mr Weiner had sent sexual messages and half-dressed photos of himself to an unidentified woman in 2015.
One image appeared to show a child next to Mr Weiner, who was in his underpants.
Mr Weiner resigned from Congress in 2011 and publicly apologised after inadvertently posting an explicit image of himself to his Twitter account.
The former congressman then launched a bid for New York City mayor in 2013, but the campaign was ultimately derailed by another sex scandal.
At the time, Ms Abedin defended the decision to remain by her husband's side, saying it was "made for me, for our son, and for our family".
Weiner, a documentary released in May, followed the politician's failed mayoral bid and his strained marriage in the wake of revelations that he had again been exchanging messages with other women.
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