The quality of post-ovulatory oocytes decreases with aging. In this study, we aimed to investigate the effects of N-acetyl-L-cysteine (NAC), a broadly used antioxidant, on oocyte quality in mouse post-ovulatory oocyte aging in vitro. NAC at 0.6mM concentration was added to culture medium (M2), and the quality of oocytes was analyzed at 6h, 12h, 18h and 24h of culture. We found that the frequency of spindle defects decreased in NAC-treated oocytes compared to those without NAC treatment. NAC treatment significantly decreased abnormal distribution of cortical granules (CGs) in oocytes during aging for 18h and 24h. Decreased intracellular reactive oxygen species (ROS) was also observed. Increased intracellular ATP levels and decreased abnormal distribution of mitochondria could be observed with NAC supplementation during post-ovulatory oocyte aging in vitro. These results indicate that NAC will maintain the quality of oocytes, and delay post-ovulatory oocyte aging as studied in the mouse.