Objective:To investigate the effects of medicated serum of Liuwei Dihuang Pill on the proliferation of MC3T3-E1 cells and mRNA expressions of Runx2 and FOXO1. MethodsThe suspension of Liuwei Dihuang Pill was prepared. The adult Wistar rats were treated with the suspension of Liuwei Dihuang Pill by intragastric administration, and then the medicated serum of Liuwei Dihuang Pill was collected. After conventional culture of MC3T3-E1 cells for 24 hours, the culture medium was replaced by 10% medicated serum with different concentrations. The proliferation of MC3T3-E1 cells was detected by CCK-8 after incubated for 48 and 72 hours respectively. The MC3T3-E1 cells were starved for 24 hours after culture with osteoblast-inducer for 22 days, and then the culture medium was replaced by 10% medicated serum with different concentrations. After cultured for 72 hours, the mRNA expressions of Runx2 and FOXO1 were detected.  Results:The medicated serum of Liuwei Dihuang Pill could promote the proliferation of MC3T3-E1 cells in a certain dose-dependent manner. Meanwhile, the mRNA expression of Runx2 was significantly increased in the medicated serum of Liuwei Dihuang Pill groups with different doses (P<0.01), and the expression in the high-dose group was significantly higher than that in the low-dose group (P<0.01). The mRNA expression of FOXO1 in the high-dose group was significantly higher than that in the other three groups (P<0.01).  Conclusion:The medicated serum of Liuwei Dihuang Pill can promote MC3T3-E1 cells proliferation and increase the mRNA expressions of Runx2 and FOXO1.