Objective：To investigate the effects of doxycycline on in vitro osteogenic differentiation of pro - osteoblasts cell line MC3T3-E1 and the possible mechanisms. Methods：Alizarin red staining was employed to detect the osteogenic differentiation. Real- time PCR was used to detect the effect of DOX on the expression of OCN and Runx2 in MC3T3-E1 cells. MC3T3-E1 cells were treated with either DOX，MEK inhibitor U0126 alone or in combination，and the levels of N-cadherin，p-MEK and p-ERK were detected by Western blot. Results：When DOX was added during osteogenic induction，alizarin red staining positivity of the cultured MC3T3-E1 was significantly enhanced. DOX increased the expression of OCN and Runx2 in MC3T3-E1 cells. Besides，DOX decreased the levels of N - cadherin and increased the level of p -MEK and p - ERK in MC3T3 - E1(P ＜ 0.05). On the contrary，MEK antagonist U0126 significantly increased the expression of N-cadherin protein and decreased the levels of p-MEK and p-ERK(P ＜ 0.05). When MC3T3-E1 cells were treated with DOX in the presence of MEK inhibitor U0126，the changes in N-cadherin，p-MEK and p-ERK were showed to be reversed comparing with DOX alone. When both U0126 and DOX were present during in vitro osteogenic differentiation of MC3T3-E1， alizarin red staining positivity was less observed than that of DOX alone. Conclusion：In this study，DOX is showed to enhance the in vitro osteogenic differentiation of MC3T3-E1，which is probably associated with MEK/ERK signaling pathway.