Abstract: MBCD and Cholesterol-Loaded-Cyclodextrin (CLC) were examined for their abilities to increase the cryosurvival of C57BL/6 mouse sperm, the main strain of genetically engineered mice. The intactness of acrosome and motility of frozen/thawed spermatozoa were used to monitor cryosurvival. In this experimental study, male mice were randomly divided in 6 groups: control 1, experimental 1, experimental 2, control 2, experimental 3 and experimental 4. In experimental groups 1 and 2 spermatozoa were exposed to 0.75 and 1 mM MBCD and in experimental groups 3 and 4 were exposed to two different concentrations of CLC (1 and 2 mg mL-1) over a period of 1 h and were subsequently cryopreserved. Spermatozoa in control 1 group were frozen without any exposure to CLC or MBCD and in control 2 (vehicle), sperms were incubated with 4 mM MBCD. The post-thaw sperms were evaluated for their motility and acrosomal status. The values of the intact acrosome and motility increased significantly with concentration of CLC compared to controls and MBCD experimental groups (p<0.05). These results indicate that cryosurvival of C57BL/6 mouse spermatozoa is enhanced by exposure to MBCD which loaded with cholesterol (CLC) before freezing and MBCD alone can not protect sperm from freeze-thaw damage efficiently compare to CLC.