Background: GDF-9 stimulates granulosa cell proliferation, and plays important roles during folliclogenesis. However, its molecular mechanisms are still far from clear, particularly its roles in human granulosa cells around the periovulatory stage. We therefore investigated the effects of GDF-9 on cell cycle distribution, regulatory molecules, and signaling pathways involved in human luteinized granulosa (hLG) cells in vitro. Methods: Primary cultures of hLG cells obtained from women undergoing IVF and treated with and without recombinant GDF-9 were evaluated with and without a specific inhibitor to ALK-5 (SB431542), ERK 42/44 (PD098059), or Smad 3 (SIS3). Cell proliferation, cell cycle distribution, and mRNA and protein expression of relevant cell cycle molecules were determined by ³H-thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. Results: GDF-9 stimulated ³H-thymidine incorporation, enhanced cell transition from G₀/G₁ to S and G₂/M phases (while both SB431542 and PD098059 attenuated these changes), increased mRNA and protein expression of cyclin D₁ and E, and decreased those of the cyclin-dependent kinase (CDK) inhibitors, p15^INK4B and p16^INK4A. GDF-9 also activated Rb protein (a critical G₁ to S-phase regulator), ERK 42/44, and Smad 3. PD098059 blocked Rb protein phorsphorylation and the increase in cyclin D₁ and E, but not the decrease in p15^INK4B and p16^INK4A induced by GDF-9. In contrast, SIS3 reversed the decrease in p15^INK4B and p16^INK4A but not the increase in cyclin D₁ and E induced by GDF-9. Conclusions: GDF-9 stimulates hLG cells proliferation by stimulating cyclin D₁ and E, and suppressing p15^INK4B and p16^INK4A via both Smad-dependent and Smad-independent pathways.