Objective To study the effects of Smad3 on the proliferation of the ovarian granulosa cells (GCS) in the rat.Methods Ovarian granulosa cells were cultured and treated in the flollowing conditions: 1. GCs were cultured without any treatment; 2. GCs were cultured with transfection reagent lipofectamine RNAiMAX; 3. GCs were transfected with transfection reagent lipofectamine RNAiMAX and Non-silencing SiRNA; 4. GCs were transfected with transfection reagent lipofectamine RNAiMAX and the SiRNA of Smad3.The expression and localization of PCNA and Cyclin D2 proteins in the four groups were detected by immuocytochemistry. Results The proteins of PCNA and Cyclin D2 were mainly located in the nuclei of GCs. The percentage of positive cells for PCNA was (25.80±1.70)% in the siRNA of Smad3 treated group, which decreased significantly compared to the control group(P <0.05),and the percentage of the stained cells for Cyclin D2 was (25.00±2.90)% in the siRNA of Smad3 treated group, which decreased significantly compared to the control group(P <0.05).Conclusion Smad3 gene could promote the proliferation of ov