This study aimed to explore the effect of sinomenine on the proliferation and differentiation of MC3T3-E1 cells and the related mechanism. Mouse preosteoblastic cell line MC3T3-E1 cells were divided into four groups: control group, treatment of sinomenine with the concentrations of 100, 500 or 1000 μM. The proliferation and apoptosis of the cells were determined by MTT assay at 0, 24, 48 and 72 h, and flow cytometry method was used to determine the effect of sinomenine on the apoptosis of MC3T3-E1 cells at 72 h; furthermore, after 72 h, the cell culture supernatants were collected, and the levels of alkaline phosphatase and osteocalcin were examined by enzyme-linked immunosorbent assay (ELISA); next, cells were collected, and the expression of type I collagen (COL1A1), osteopontin (OPN), protein kinase B (Akt), runt-related transcription factor 2 (Runx2), B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were examined by RT-qPCR and Western Blot methods. It was observed that treatment of 500 and 1000 μM sinomenine has induced significant increase in the proliferation and decrease in the apoptosis of MC3T3-E1 cells; furthermore, sinomenine also lead to increased secretion of osteocalcin and alkaline phosphatase in MC3T3-E1 cells; finally, sinomenine induced marked increase in the expression of type I collagen, osteopontin and also induced the activation of the Akt/Runx2 signaling pathway. To sum up, we observed that sinomenine may promote the proliferation and differentiation of MC3T3-E1 cells via activating the Akt/Runx2 signaling pathway.