Luteinization is a vital process in female reproduction, where granulosa cells differentiate into progesterone-secreting luteal cells. While luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are established promoters of luteinization, the regulatory role of activin A remains incompletely understood. This study aimed to investigate whether activin A can prevent or reverse luteinization in granulosa cells from small antral and preovulatory follicles using an in vitro model. Granulosa cells were collected from women undergoing either IVF treatment or ovarian tissue cryopreservation. Cells were cultured for 48 h with FSH, hCG, or activin A. Hormone secretion (progesterone and estradiol) and content of steroidogenic markers (CYP19A1, STAR, 17βHSD1, 3βHSD2, FSHR, and LHCGR) were analyzed via ELISA, RT-qPCR, and Western blot. RESULTS: demonstrated that activin A suppressed luteinization markers and progesterone production while promoting estrogen synthesis and maintaining granulosa cell features. Conversely, hCG increased luteinization marker expression and progesterone secretion, consistent with its known role in luteal transformation. Notably, granulosa cells from small antral follicles showed a distinct, stage-specific response to activin A compared to those from preovulatory follicles, indicating a temporal regulation of luteinization competence. These findings support a regulatory role for activin A in maintaining granulosa cell identity and delaying luteinization. Understanding the differential responsiveness of granulosa cells across follicular development stages may inform new therapeutic approaches for luteal phase deficiencies and fertility preservation strategies.