Objective To investigate the inhibitory effect of Pingliukang (a prescription) medicated serum on the proliferation of cultured rat C6 glioma cells and influence on cell cycle in vitro. Methods MTT colorimetry were performed to measure the levels of the proliferation of rat C6 glioma cells cultured with 2.5%, 5%, 10% and 20% of Pingliukang medicated serum for 24 h, 48 h and 72 h in vitro. The effect of Pingliukang medicated serum on cell cycle were observed by FCM. Results The proliferation of C6 cell was obviously inhibited by Pingliukang medicated serum with dose-effect relationship. The inhibitory effect of 20% of medicated serum was the strongest. When the C6 glioma cells were treated with 10% and 20% medicated serum for 48 h and 72 h, the cells of S period declined. Conclusion The Pingliukang medicated serum can inhibit the proliferation and block cell cycle of cultured C6 glioma cells.