Background: N-acetylcysteine (NAC) is a supplement commonly used in patients with polycystic ovary syndrome (PCOS). The expansion of oocyte-associated cumulus cells (CCs) and the quality of the oocyte are critical factors influencing fertilization rates and clinical pregnancy outcomes in assisted reproductive techniques (ARTs). Genes such as phosphofructokinase (PFKP) and pyruvate kinase isoform M2 (PKM2) are involved in glucose metabolism and are crucial in the regulation of oocyte competence and developmental potential. The purpose of the current study was to evaluate the effects of letrozole and NAC on the expression of PFKP and PKM2 in CCs of PCOS patients undergoing ART. Methods: The study evaluated 20 PCOS women undergoing ART to assess the effect of letrozole and NAC on the expression levels of PKM2 and PFKP genes in cumulus cells. Women were randomly assigned using a simple randomization method into four groups: control, NAC, letrozole, and NAC plus letrozole, with five women in each group. Gene expression levels of PKM2 and PFKP were measured using real-time PCR. Results: The expression level of PKM2 was significantly higher in the letrozole plus NAC group compared to the control group (p<0.05). In NAC group, PFKP was significantly expressed compared to the control group (p<0.05). There were no significant differences among the other groups compared to the control group. Conclusion: NAC can improve the quality of oocytes by increasing the expression level of genes involved in the glucose metabolism (PKM2, PFKP) of CCs, thereby potentially improving ART success rate in PCOS patients. Therefore, administering NAC along with letrozole can have a synergistic effect on increasing the expression level of genes associated with blastocyst quality in PCOS patients.