The new Ryanair route, to Knock airport, will be up and running from summer next year.
There will be three flights per week on Wednesdays, Fridays and Sundays.
Mayo is Ireland's third biggest county and its western coastline boasts rugged mountain trails and beautiful beaches.
It also includes Achill, Ireland's largest island, which is accessible from the mainland by bridge.
The area is also famous for the Knock Shrine, one of Europe's major Marian shrines.
One and a half million pilgrims visit it annually and Pope John Paul II was there in 1979 to commemorate its centenary celebrations.
Prestwick's Paula Horne said: "It's fantastic to have a new name on our departure board for next spring and summer and it's also a great vote of confidence by Ryanair in the airport's future.
"Scots will love the County Mayo area and it's just a short flight away from Prestwick."