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Objective:To optimize the cryopreservation of buffalo bull semen by altering freezing rates within critical temperature range (4℃ to -60℃). Methods: A total of 20 ejaculates each from 5 Murrah buffalo bulls were cryopreserved using programmable biofreezer in 2 phases. In the 1st phase, 9 freezing rates were applied at -2, -5, -10, -20, -30, -40, -50, -60 or -4℃/min (control) from 4℃ to -15℃; at -40℃/min from -15℃ to -60℃. In the 2nd phase, a fixed freezing rate was applied at -30℃/min from 4℃ to -15℃. Six freezing rates were applied at -10, -20, -30, -40 (control), -50 or -60℃/min from -15℃ to -60℃. The freezing from -60℃ to -140℃ were fixed at -50℃/min in both the phases. Post thaw semen quality was assessed in terms of motility, viability, membrane integrity (hypo-osmotic swelling test), sperm abnormalities, and active mitochondria. Data were arc sine transformed and analyzed through one-way analysis of variance using SPSS software. Results: In the 1st phase, percent individual motility, progressive motility and viability were similar among various protocols. Percent hypo-osmotic swelling reactive sperm was higher with freezing at -30℃/min. In the 2nd phase, percent individual motility, viability and hypo-osmotic swelling reactive sperm was higher with freezing at -50℃/min. Sperm head abnormalities were lower at -30℃/min in the 1st phase, but were similar among the protocols of the 2nd phase. Percent active mitochondria were higher at -30℃/min in the 1st phase and at -50℃/min in the 2nd phase.Conclusions:The optimum post thaw semen quality of buffalo bull could be obtained by applying freezing rate at -30℃/min (4℃ to -15℃) and at -50℃/min (-15℃ to -140℃), followed by plunging of straws in into liquid nitrogen for storage.