Basal generation of reactive oxygen species (ROS) was essential for male reproductive function, whereas high ROS levels may be linked to low quality of sperm and male infertility. We examined the associations between ROS levels in whole ejaculates and sperm quality among 1092 male factor infertility (MFI) patients and 50 donors with normal semen characteristics. ROS levels were significantly positively correlated with abnormal morphology rate, head defect, and sperm deformity index. Further, we investigated whether seminal plasma from MFI patients with high ROS levels affects sperm motility from donors with normal semen characteristics. After cross-culturing fresh human sperm from donors possessing normal semen characteristics with seminal plasma from infertitle men, sperm motility was measured at different ROS levels. Seminal plasma from MFI patients significantly reduced motility of sperm and the reduction rate increased with increasing ROS levels in seminal plasma. On the other hand, we found MFI patients with the ROS levels in the lowest 25th percentile had similar ROS levels to donors with normal semen characteristics. Collectively, our observations lead to the hypothesis that oxidative stress plays a critical role in the development of MFI among those with high ROS levels, but not those with low ROS levels. Antioxid. Redox Signal. 16, 754-759.