The study was carried out to understand the effect of various concentrations of cholesterol loaded cyclodextrin (CLC) on cryosurvivability of buffalo spermatozoa. Twenty four ejaculates with mass motility GT= 3+ from 4 bulls (6 from each bull) were collected as per standard procedure. Each ejaculate was split into four groups viz., Group I (control: extended with Tris-egg yolk-glycerol extender upto 80*106 sperm/ml), Group II (treated with CLC 1 mg/ml), Group III (treated with CLC 2 mg/ml) and Group IV (treated with CLC 3 mg/ml). Semen samples of Group II, III or IV were incubated with CLC at 37 deg C for 15 minutes for the entry of cholesterol into sperms and finally diluted upto 80*106 sperm/ml. The results revealed that post-dilution and after freezing at 0.7 and 30 days of cryopreservation motility, live and dead, acrosomal integrity and hypo-osmotic swelling test were significantly (P LT 0.05) higher in Group III as compared to all other groups. However, significantly (P LT 0.05) higher percentage of abnormal sperm was observed in control group as compared to other groups. Therefore, addition of CLC 2 mg/ml in extender during buffalo semen cryopreservation has beneficial role in improvement of semen quality.