The study was conducted to investigate the effects of different levels of cholesterol loaded cyclodextrin (CLC) addition on cooled and frozen-thawed spermatozoa of Marwari stallion. A total of 48 ejaculates were collected from six adult Marwari stallions (8 ejaculates from each stallion) aged between 4 to 7 years. Immediately after collection semen sample was macroscopically evaluated and filtered into a warm, graduated measuring bottle to get gel free semen. The level of cholesterol (C) and phospholipid (P) in fresh spermatozoa were measured using ELISA. The semen sample was divided in to five equal aliquots (T0, T1, T2, T3 and T4). Primary extender containing different concentrations of CLC was added to each aliquot (0, 1,... 1.5, 2 and 3 mg/ml CLC in T0, T1, T2, T3 and T4, respectively). All the aliquots were incubated for 15 minutes at 37°C for incorporation of CLC in sperm plasma membrane and then cryopreserved. Level of C and P in spermatozoa was also evaluated at pre-freeze and post-thaw stages. The mean C, P and C: P ratio in fresh sperm was 15.36±0.47 μg/100 × 106 sperm cells, 46.21±1.27 μg/100 × 106 sperm cells and 0.33±0.071, respectively. The mean C content and C: P ratio were significantly higher (P<0.05) in group T3 as compared to other groups at both pre-freeze and post-thaw stages. It was concluded that addition of CLC may be helpful in increasing cryopservability of stallion spermatozoa.