Objective To explore the mechanism of the rat hyperplasia suppressor gene (rHSG) inhibited proliferation in C6 rat glioma cells. Methods C6 cells were cultured in vitro and transduced with the adenovirus vector which carried rHSG gene (Adv-rHSG-GFP). The transduction efficiency of adenovirus vector was measured by inverted microscope and flow cytometry in C6 cells. Flow cytometry was used to analyze the C6 cells cycle. Western blot was adopted to test the change of rHSG protein expression, the protein of cancer suppressor gene P53, cell cycle control protein of P21cip1, phosphorylation and non-phosphorylation retinoblastoma proteins (p-Rb, Rb). Results The adenovirus can insert the target gene into the genome of C6 target cell efficiently. The expression level of rHSG protein of Adv-rHSG-GFP group is obviously higher than that of PBS Group and Adv-GFP group. Meanwhile, the over-expressed C6 cells of rHSG that arrest in G0/G1 phase are largely increased (P < 0.01). Besides, there is a large increase in the protein expression of P53 and P21cip1 (P < 0.01), decrease in the expression of p-Rb (P < 0.01) and no significant change in the expression of Rb (P < 0.05). Conclusion rHSG might inhibit the proliferation of C6 rat glioma cells through P53-P21cip1 pathway.