Abstract The present study was undertaken to evaluate antioxidant’s vitamins concentrations in serum, follicular fluid and corpus luteum of cyclic buffalo cows. A total of 34 clinically healthy buffalo cows (Bubalus bubalis), aged 8–10 years, were subjected to study. All animals were examined before slaughtering and the findings on the ovaries and the uterus were recorded. Blood samples and the whole genital tract of each animal were collected just after slaughtering. Antioxidants were measured in serum, CL and follicular fluid at different stage of the estrus cycle (proestrus n08, estrus n07, metestrus n07, and diestrus n012). The results revealed significant increases in serum α-tocopherol concentration during metestrus and diestrus stages (p0.05). On the other hand, there was a significant decrease (p0.05) in follicular ascorbic acid concentration at diestrus phase. Follicular β-carotene (p0.01) showed a significant increase at the metestrus than at estrus and diestrus phases of the cycle. Follicular Vitamin A significantly increased (p0.01) at proestrus phase. Although during metestrus there was significant decrease in the corpus hemorrhagicum weight (p0.01), ascorbic acid concentration was significantly increased (p0.05). In conclusion, the results of the present study suggested that serum α-tocopherol concentration increased during metestrus and diestrus stages, follicular vitamin A increased in proestrus phases when regeneration and steroideogenesis are required. Ascorbic acid increased in diestrus phase to help corpus luteum to function properly.