Objective: To assess the effect of catalase (CAT) on sperm motility, viability, total sperm abnormality, acrosomal and plasma membrane integrity, enzymatic profiles such as aspartate amino transaminase (AST), alanine amino transaminase (ALT), biochemical profiles such as cholesterol efflux and malonaldehyde (MDA) production and antioxidant profiles such as reduced glutathione (GSH), superoxide dismutase (SOD) and total antioxidant capacity (TAC). Methods: Total numbers of 50 ejaculates were collected twice a week from eight mithun bulls and semen was split into four equal aliquots, diluted with the TEYC extender. Group 1: semen without additives (control), group 2 to group 4: semen was diluted with 50 U/mL, 100 U/mL and 150 U/mL of catalase, respectively. These seminal, enzymatic, biochemical and antioxidant profiles were assessed at 5 ℃ for 0, 6, 12, 24 and 30 h of incubation. Results: Inclusion of catalase into diluent resulted in significant (P < 0.05) decrease in percentages of dead spermatozoa, abnormal spermatozoa and acrosomal abnormalities at different hours of storage periods as compared with control group. Additionally, CAT at 50 and 150 U/mL were inferior to CAT 100 U/mL treatments as regards to these characteristics and CAT at 100 U/mL has significant improvement in quality of mithun semen in in- vitro stored for up to 30 h.Conclusions:It was concluded that the possible protective effects of CAT on sperm parameters are it prevent efflux of cholesterol from cell membrane, MDA production and protect the function of antioxidants during preservation.