The pair announced the news "with great sadness"  in a statement on Twitter, but said they remained "great friends".
"After many exciting adventures together over the last 18 years we have come to the end of our rainbow," the pair said.
BBC Radio 2 DJ Ball and Cook married in 1999 and have two children together.
In the statement, they said: "We are still great friends and will continue to support each other and raise our beautiful children together, living next door but one."
The couple, who live in Brighton, East Sussex, had their first child, Woody, in 2000.
They announced they were to separate in 2003, but reconciled, and had their second child, Nelly, in 2010.
Ball, 45, is best known as a former BBC Radio 1 breakfast show host and currently hosts Strictly Come Dancing: It Takes Two.
Cook, 53, is a former member of the band the Housemartins and under his pseudonym Fatboy Slim enjoyed success with songs including Praise You; Right Here, Right Now; and Weapon of Choice.
TV presenter Zoe Ball and husband DJ Norman Cook - better known as Fatboy Slim - have announced their separation after 18 years together.