Kix Brooks and Ronnie Dunn, who will launch a farewell tour next year, released a statement on Monday explaining the decision.
It read: "After 20 years of making music and riding this trail together, we have agreed as a duo that it's time call it a day.
"This ride has been everything and more than we could ever have dreamed.... We owe it all to you, the fans. If you hear rumours, don't believe them, it's just time."
Brooks and Dunn have sold more than 30 million albums and won two Grammy Awards. They are best known for tracks such as 'Brand New Man' and 'Only in America'.