Abstract: Objective To investigate the role of miR-144-3p in regulating osteogenic differentiation of bone marrow mesenchymal stem cells and predict its target genes. Methods Rat bone marrow mesenchymal stem cells (BMSCs) with induced osteogenic differentiation were examined for the expressions of Runx2, OCN and miR-144-3p. The effects of transfection with a miR-144-3p mimic or a miR-144-3p inhibitor were tested on the osteogenic differentiation of the BMSCs. The changes in the expressions of the predicted target of miR-144-3p in the BMSCs during induced osteogenic differentiation were examined using Western blotting and qRT-PCR. Results Rat BMSCs with induced differentiation into osteoblasts exhibited a progressive increase in the expressions of Runx2 and OCN (two markers of osteogenic differentiation), while the expression of miR-144-3p gradually decreased during the differentiation till reaching the lowest level at 21 days of induction. In rat BMSCs, transfection with the miR-144-3p mimic significantly decreased ALP activity (P<0.05) wile transfection with the miR-144-3p inhibitor significantly increased ALP activity (P<0.05) in rat BMSCs. Analysis based on miRanda, microRNA.org database and TargetScan suggested that Smad4 was the most likely target gene of miR-144-3p. The results of qRT-PCR showed no significant differences in expression levels of Smad4 among the cells with different treatments (P>0.05), while Western blotting revealed a significantly decreased expression of Smad4 in the cells transfected with miR-144-3p mimics and an increased Smad4 expression in the cells transfected with the miR-144-3p inhibitor as compared with the control cells (P<0.05). Conclusion miR-144-3p participates in the regulation of osteogenic differentiation of rat BMSCs, and its inhibitory effect on osteogenic differentiation is achieved probably by decreasing the expression level of Smad4.