The aim of the present study was to investigate the effect of in vitro supplementation of selenium on fresh and frozen spermatozoa quality of buffalo (Bubalus bubalis) bulls. Five healthy buffalo bulls (5 ejaculates from each bull) were used. Each ejaculate was diluted at 37 ˚C with tris-based extender containing 0 (control), 0.5, 1, 2, 4 and 8 μg mL-1 sodium selenite and the sperm motility and viability were evaluated at 0 (T0) (immediately after dilution), 60 (T1) and 120 (T2) min after diluting semen. In the second step, semen samples were diluted with tris-egg yolk-glycerol extender containing the same amounts of sodium selenite, cooled to 4 ˚C, equilibrated and semen parameters (motility, viability, membrane integrity and DNA damage) were estimated. Then, the semen was packed in 0.5 mL French straws and frozen in liquid nitrogen. Later, the semen was thawed and analyzed for the same parameters, as well as total antioxidant capacity. Results showed that addition of 1 and 2 μgmL-1 selenium to the semen extender significantly increased the sperm motility of fresh and equilibrated semen compared to the control without affecting other parameters. However, in frozen-thawed semen, extenders containing 1 and 2 μg mL-1 selenium significantly improved sperm motility, viability, membrane integrity and semen total antioxidant capacity and also resulted in lower DNA damaged sperms. In this study selenium supplementation of semen extender of 4 and 8 μg mL-1 had deleterious effects on sperm parameters as early as the samples were prepared for freezing.