BACKGROUND Mesenchymal stem cells (MSCs) are drawing considerable attention in the field of regenerative medicine due to their differentiation capabilities. The miRNAs are among the most important epigenetic regulators of MSC differentiation. Our previous study identified miR-4699 as a direct suppressor of the DKK1 and TNSF11 gene expression. However, the precise osteogenic-related phenotype or mechanism caused by miR-4699 change has yet to be dealt with in depth. METHODS In the present study, miR-4699 mimics were transfected into human Adipose tissue-derived mesenchymal stem cells (hAd-MSCs) and osteoblast marker gene expression (RUNX2, ALP, and OCN), was analyzed to investigate whether miR-4699 promotes osteoblast differentiation of hAd-MSCs through targeting the DKK-1 and TNFSF11. We further examined and compared the effects of recombinant human BMP2 with miR-4699 on cell differentiation. In addition to quantitative PCR, analysis of alkaline phosphatase activity, calcium content assay, and Alizarin red staining were used to explore osteogenic differentiation. To evaluate the effect of miR-4699 on its target gene (on protein level) we utilized the western blotting technique. RESULTS The overexpression of miR-4699 in hAd-MSCs resulted in the stimulation of alkaline phosphatase activity, osteoblast mineralization, and the expression of RUNX2, ALP, and OCN osteoblast marker genes. CONCLUSIONS Our findings indicated that miR-4699 supported and synergized the BMP2-induced osteoblast differentiation of mesenchymal stem cells. We suggest, thereof, the utilization of hsa-miR-4699 for further in vivo experimental investigation to reveal the potential therapeutic impact of regenerative medicine for different types of bone defects.