The purpose of this study was to examine the effects of sub-lethal concentration of Xanthine oxidase (XO) on the post-thawed bull sperm quality. Semen samples were collected from four Holstein bulls, twice a week and during three consecutive weeks (n = 24 total ejaculates). After collection in each replicate, semen samples were pooled and then frozen by semen extender containing different concentrations [0 (XO-0), 0.05 (XO-0.05), 0.5 (XO-0.5), 5 (XO-5), 50 (XO-50) and 500 (XO-500) μM] of XO. After thawing, motion parameters (SCA), plasma membrane functionality (HOST), apoptosis status (Phosphatidylserine translocation assay), mitochondrial activity (Rhodamine 123), and acrosome integrity (PSA), were evaluated. The results showed that total motility, VAP, VSL, VCL, STR, and LIN were lower in XO-50 and XO-500 compared to other groups (P < 0.05). Progressive motility were higher in XO-0.05 and XO-0.5 compared to XO-0, XO-50, and XO-500 (P < 0.05). Mitochondrial activity was highest in XO-0.05 and XO-0.5 groups. Sperm plasma membrane functionality was significantly greater in XO-0, XO-0.05, XO-0.5, and XO-5 than that of XO-50 and XO-500. Xanthine oxidase had not significant effects on acrosome integrity and dead spermatozoa. Higher percentage of live spermatozoa was recorded for XO-0, XO-0.05, XO-0.5, and XO-5; however, the lower amount of apoptotic spermatozoa was detected in the aforementioned groups (P < 0.05). In conclusion, it seems that XO at lower doses may have beneficial effects on post-thawed bull sperm quality.