Objective To investigate the effects of Shikonin on proliferation and osteogenic differentiation of mouse preosteoblast cells（MC3T3⁃E1 cells）and the role of OPG/ RANKL signaling axis. Methods MC3T3⁃E1 cells were cultured in vitro and divided into control group and different concentrations of shikonin group（0.0625， 0.125，0.25，0.5，1，2 μmol/L）. After 24，48，72 h of administration，Cell proliferation inhibition rate was detected by CCK⁃8 method；ALP activity of MC3T3⁃E1 cells was detected by alkaline phosphatase（ALP）activity kit. The NBT/BCIP kit was used to identify ALP staining. The expression levels of osteoprotegerin（OPG），nuclear factor κB receptor activating factor ligand（RANKL），Runt ⁃associated transcription factor 2（RUNX2）and type ⁃ I collagen （COL ⁃ I）genes were detected by Real ⁃time fluorescence quantitative PCR（RT ⁃PCR）；Alizarin red staining was used to observe the formation of mineralized nodules. Results Compared with the control group，0.0625，0.125， 0.25 and 0.5 μmol/L shikonin groups significantly promoted the proliferation of MC3T3⁃E1 cells（P < 0.05），while 0.5 μmol/L shikonin group significantly inhibited the growth of MC3T3⁃E1 cells（P < 0.05）. The safe concentration of shikonin group promoted the proliferation，differentiation and mineralization of osteoblasts in a concentration⁃ dependent manner. RT⁃PCR results showed that，when compared to the control group，the safe Shikonin concentra⁃ tion significantly increased the expression of OPG，RUNX2，and COL⁃I while inhibiting the expression of RANKL （P < 0.05）. Conclusions Shikonin can promote the proliferation，differentiation and mineralization of MC3T3⁃E1 cells，possibly by regulating the OPG/ RANKL signaling axis and osteogenic genes.