Objective In this study, the molecular mechanism by which microRNA?494 inhibits osteoblast differentiation and matrix mineralization through the TLR?4 pathway was examined. Methods The mouse osteoblast strain MC3T3?E1 was selected as an in vitro model, which was transfected with miR?494 mimic. The expression of miR?494 before and after this transfection was determined by qRT?PCR. The proliferation of MC3T3?E1 before and after transfection was also detected by MTT assay, while the differentiation was studied by measuring ALP activity. In addition, matrix mineralization of this cell line was determined by quantitative detection of osteocalcin (OC) and Von Kossa calcification staining. Finally, the difference of TLR?4 protein expression before and after transfection was determined by Western blotting. Results Compared with that in the negative control group, the mRNA expression of miR?494 after transfection of the mouse osteoblast cell line MC3T3?E1 was significantly increased ( P < 0. 05). The result showed that miR?494 mimic was transfected into this cell line and successfully led to the overexpression of miR?494. Compared with the negative control group, after transfection with miR?494 mimic, the proliferative capacity of mouse MC3T3?E1 osteoblasts was inhibited ( P < 0. 05). ALP activity in MC3T3?E1 was also significantly decreased after transfection ( P < 0. 05), indicating that the differentiation ability of MC3T3?E1 cells was inhibited. At the same time, the activity of OC in MC3T3?E1 was significantly decreased after transfection ( P < 0. 05) and the number of mineralized nodules was also significantly decreased ( P <0. 05), which indicated that the overexpression of miR?494 inhibited the mineralization of matrix in these cells. Western blot result showed that TLR?4 protein expression was significantly increased in cells transfected with miR?494 mimic, compared with that in the negative control group ( P < 0. 05). Conclusions miR?494 could inhibit the proliferative activity of the mouse osteoblast line MC3T3?E1 through the TLR?4 pathway, reduce the activity of ALP and OC in cells, decrease the number of mineralized nodules in osteoblasts, and inhibit osteoblast differentiation and matrix mineralization.