The Dutch Tweede Divisie in the 1959–60 season was contested by 25 teams, twelve of which playing in group A, thirteen in group B.
Eight teams would play against relegation this season in a play-off. Two teams already had to play in it on the basis of last season's results: ONA and Velocitas 1897. The other six participants were the lowest ranked teams of this season. The changes were part of an attempt by the KNVB to make the Tweede Divisie one league next year. Also, one extra team would be promoted to the Eerste Divisie this year, through a play-off between the third-place finishers of both groups.
EBOH were promoted to Eerste Divisie.
Enschedese Boys were promoted to Eerste Divisie.
The two losing sides played off for the final promotion spot:
sc Heerenveen were promoted to Eerste Divisie.