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.