Objective To study the effect of continuous tensile stress on expression of Forkhead box protein O1 (FoxO1) in MC3T3-E1 cells in vitro during osteogenic differentiation, and explore the role of FoxO1 in the mechanism of continuous tensile stress induced-osteogenic differentiation. Methods MC3T3-E1 cells were seeded and applied with tensile stress at the frequency of 1 Hz and amplitude of 10% by FX-4000TTM mechanical loading system. MC3T3-E1 cells were divided into control, 1 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h group, respectively, according to the time subjected to the tensile stress. Alkaline phosphatase (ALP) staining, real-time PCR, Western blotting and immunofluorescence were applied to detect the effects of continuous tensile stress on osteogenesis ability of MC3T3-E1 cells, mRNA and protein expression of FoxO1, and allocation of FoxO1 in MC3T3-E1 cells. Results (1) Continuous tensile stress could promote the osteogenic differentiation of MC3T3-E1 cells. Compared with the control group, the mRNA expression of ALP increased significantly at 24 h, 48 h, and the mRNA expression level of osteocalcin (OCN) reached the peak value at 72 h, which was significantly higher than that in the control group. The mRNA expression of runt-related transcription factor-2 (Runx2) significantly increased at 4 h as compared to the control group, and Runx2 protein level changed accordingly. The ALP staining results of the stress group and control group were significantly different. (2) Continuous tensile stress could increase mRNA and protein expression of FoxO1. The mRNA expression of FoxO1 markedly increased at 24 h, and its protein expression significantly elevated at 12 h. (3) FoxO1 was expressed in the nucleus and cytoplasm at 6 h, and then significantly increased in the cytoplasmat at 24 h. Conclusions 10% continuous tensile stress can stimulate the osteogenic differentiation of MC3T3-E1 cells, up-regulate the mRNA and protein expression of FoxO1 and change the allocation of FoxO1 in MC3T3-E1 cells. The investigation on the change rules of FoxO1 expression and allocation under mechanical stimulation will provide the experimental basis for studying the role of FoxO1 in mechanical stimulation.