The present study was undertaken to assess the effect of trehalose on sperm parameters, enzymatic profiles and biochemical profiles of seminal plasma of mithun. The semen ejaculates were collected from mithun and was split into four groups as group 1: semen without additives (control), group 2 to 4: semen with 50 mM, 75 mM and 100 mM of trehalose, respectively in EYTC extender. These parameters were assessed at 5°C for 0, 6, 12, 24 and 30 h of incubation. Inclusion of trehalose resulted in significant (p < 0.05) decrease in percentages of dead spermatozoa, abnormal spermatozoa and acrosomal abnormalities as compared with control group. Additionally, trehalose at 50 mM has significant improvement in quality of mithun semen in in- vitro storage. It was due to the protective effects of trehalose on sperm parameters as sperm membrane stabilizer and prevents efflux of cholesterol, lipid peroxide formation and biochemical enzymes from sperm cell during preservation. [ABSTRACT FROM AUTHOR]