The purpose of this study was to investigate the role of Icariine on the expression of Smadl and Smad5 mRNA and protein levels in MC3T3-E1 cells in vitro. MC3T3-E1 cells were cultured in the presence of different concentrations of Icariine (0, 10, 40 and 80 ng/ml). Smad1 and Smad5 mRNA levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and the expression of proteins was determined by western blotting, immunohistochemistry staining and immunofluorescence. Smad1 and Smad5 mRNA levels continuously increased in 10, 40 and 80 ng/ml of Icariine with time and the differences indicated statistical significance. Western blot analysis demonstrated that the Smad1 and Smad5 protein levels in the 10, 40 and 80 ng/ml groups were higher compared with the 0 ng/ml group at 24, 48 and 72 h, and the difference was statistically significant. Immunohistochemistry staining and immunofluorescence showed that the expression of the Smad1 and Smad5 proteins was higher in the cytoplasm and nuclei in the 10, 40 and 80 ng/ml groups compared with the 0 ng/ml group. Icariine has a direct stimulatory function on the differentiation of MC3T3-E1 osteoblastic cells in vitro, which may be mediated by increasing the production of Smad1 and Smad5 in osteoblasts.