Semen was collected twice daily (100 ejaculates) from 5 sexually mature buffalo bulls (7-10 y old and 550-620 kg LBW) during the interval from January to April 2007 to study the effect of different types and levels of antioxidants on sperm motility and acrosome status in post-thawed buffalo semen. The main extender used for semen dilution was Tris-egg-yolk-citrate containing 7% glycerol. Total of 10 extenders, 9 with 3 types and 3 levels of antioxidants, including catalase (250, 500 and 1000 IU), glutathione (GSH, 0.4, 0.8 and 1.2 mM) and ascorbic acid (AA, 0.5, 1.0 and 1.5 g/l) were compared to unsupplemented extender (control). Semen was extended with different extenders, frozen in Liquid nitrogen (-196^oC) and thawed at rates of 15/60, 35/30 and 55/15 ^oC/sec.  Percentages of sperm motility and damage acrosome were determined in post-thawed semen. Conception rate was detected in 100 sexually mature buffalo cows.  Results revealed that adding all antioxidants to semen extenders increased percentage of sperm motility compared with the control, except for the highest level of AA (1.5 g/l), which did not affect sperm motility percentage. The best improvement was catalase at a level of 1000 IU (62.1%). Adding all levels of catalase and the lowest level of GSH (0.4 mM) or AA (0.5 g/l) decreased percentage of sperm with damage acrosome as compared to the control. The highest level of catalase (1000 IU) showed the highest impact on reducing percentage of sperm with damage acrosome (6.0%). Sperm motility percentage was higher with a thawed rate of 55/15 than 35/30 than 15^oC/60 sec, being 43.0, 32.11 and 27.0%, respectively). Adding catalase (1000 IU) or AA (0.5 or 1.0 g/l) increased sperm motility percentage in semen thawed by different rates, being 38.3, 37.4 and 37.1%, respectively. The highest conception rate (80%) was obtained from buffalo cows inseminated with semen extended using catalase (1000 IU). In conclusion, Tris-based extender containing catalase at a level of 1000 IU in frozen semen thawed at a rate of 55^oC/15 sec showed the highest post-thawing motility and the best fertilizing capacity of buffalo spermatozoa.