The purpose of this study was to investigate the effects of rosiglitazone on ram semen after cryopreservation on the quality of thawed sperm. Sperm motility, membrane functionality, viability, total abnormality, acrosome membrane integrity, mitochondrial activity, reactive oxygen species production, ATP content and apoptotic features were assessed after thawing. Rosiglitazone at concentration of 60 μM resulted in the highest (P < 0.05) total motility, progressive motility and straight-line velocity. The percentages of average path velocity and curvilinear velocity were greater in the 60 μM group. Different concentrations of rosiglitazone did not have significant effects on amplitude of the lateral head displacement, linearity and straightness. The highest amounts of membrane functionality and mitochondrial activity after freeze-thawing were observed in groups containing 60 μM. By increasing the rosiglitazone level to 80 μM, no positive effect was observed in most of the evaluated parameters. The lowest ROS concentration was recorded in 60 μM rosiglitazone group (P < 0.05). The group containing 60 μM rosiglitazone also produced the lowest significant percentage of apoptosis-like changes and dead sperm. A greater (P < 0.05) percentage of acrosome integrity in frozen-thawed spermatozoa was observed in the 60 μM rosiglitazone group. There was no significant difference between 40 and 60 μM rosiglitazone in intact acrosome of ram thawed semen. The result showed that supplementation in ram semen extender with rosiglitazone had a positive role in the regulation of ram sperm motility and had strong protective effect on the sperm membrane and acrosome integrity.