Present study was designed to ascertain antioxidative enzyme shift during cryopreservation of buffalo bull spermatozoa. Enzymatic profile of three enzymes (catalase, superoxide dismutase and glutathione peroxidase/reductase) was established for fresh, post-dilution, pre-freezing and post-thaw stages. A total of twenty semen samples from four bulls were cryopreserved in French straws by using tris based extender. Catalase (U/mL), superoxide dismutase (U/mL) and total glutathione (µM) profile for fresh semen (25.8±1.6, 93.1±1.3, 77.4±5.2; respectively), lowered with semen extension (6.0±0.6, 60.7±2.4, 63.0±0.9 respectively; P<0.05).During equilibration time, the enzymatic profile for catalase remained stable (7.0±0.7; P>0.05) but superoxide dismutase and glutathione profile deteriorated further (47.7±1.4, 44.6±0.6 respectively; P<0.05). During cryopreservation, major shift was observed in catalase profile (2.3±0.3; P<0.05) while superoxide dismutase and glutathione readings were stable in post-thaw evaluations (P>0.05). It is concluded that cryopreservation process deteriorated catalase enzyme profile while superoxide dismutase and glutathione profiles are declined by metabolic activities of spermatozoa in time dependent manner.