Abstract: Objective To investigate the effects of smokeless tobacco extract (STE) on biological properties of osteoblast, and to identify possible pathological mechanisms of osseointegration. Methods MC3T3-E1 Sub-clone 14 cells were cultured in the presence of STE at 0 （control group）,0.01,0.1,1,5,10 g/L. The cell proliferation was measured by MTT assay 1 d, 3 d, 5 d, and 7 d after exposure. The F-actin cytoskeleton of MC3T3 was stained with Rhodamine and DAPI, and then examined under a confocal laser scanning microscope 24 h after exposure to STE. The mRNA expressions of interleukin-6 (IL-6) and core-binding factor α1(Cbfα1) were quantified by real-time PCR (RT-qPCR) 48 h after exposure to STE. Results The MTT assay showed that 0.01-10 g/L STE inhibited MC3T3 proliferation (P<0.05). Prolonged time enabled 5-10 g/L STE to inhibit MC3T3 proliferation (P<0.05). Network structure in F-actin cytoskeleton was demonstrated in the controls. In the cells exposed to STE, F-actin cytoskeleton started to change with disruptive structures. As the concentration of STE increased, the changes became more significant. STE increased the mRNA expression of IL-6 at the concentration of 5 g/L and 10 g/L (P<0.05), decreased the mRNA expression of Cbfα1 at the concentration of 0.1-10 g/L (P<0.05). Conclusion Tobacco may inhibit osteoblast proliferation, destroy F-actin cytoskeleton structure, increase the mRNA expression of IL-6 and decrease the mRNA expression of Cbfα1, and inhibit cell differentiation and adhesion accordingly. Smoking is a disadvantage to osseointegration.