--- - |2 Abstract Background Osteoblastic differentiation of mesenchymal stem cells (MSCs) is the principal stage during the restoration and regeneration of bone tissue. Epigenetic modifications such as DNA methylation play a key role in the differentiation process of stem cells. In this study, the methylation status of the promoter region of ZBTB16 and Twist1 genes and their role in controlling osteoblastic differentiation in MSCs was investigated during the osteoblastic differentiation of MSCs. Methods The MSCs were cultured under standard conditions and differentiated into the osteoblasts. We had three treatment groups including 5‐azacytidine (methylation inhibitor), metformin (Twist‐inhibitor), and procaine (Wnt/β‐catenin inhibitor) and a non‐treated group (control). Methylation level of DNA in the promoter regions was monitored by methylation specific‐quantitative polymerase chain reaction (PCR). Also, the mRNA levels of key genes in osteoblastic differentiation were measured using real‐time PCR. Results ZBTB16 gene expression was upregulated, and promoter methylation was decreased. For Twist1 messenger RNA (mRNA) level decreased and promoter methylation increased during osteoblastic differentiation of MSCs. 5‐Azacytidine caused a significant reduction in methylation and increased the mRNA expression of ZBTB16 and Twist1. Metformin repressed the Twist1 expression, and therefore osteoblastic differentiation was increased. On the opposite side, procaine could block the WNT/β‐catenin signaling pathway, as a consequence the gene expression of key genes involved in osteoblastic differentiation was declined. Conclusion We found that methylation of DNA in the promoter region of ZBTB16 and Twist1 genes might be one of the main mechanisms that controlling the gene expression during osteoblastic differentiation of MSCs. Also, we could find an association between regulation of Twist1 and ZBTB16 genes and osteoblastic differentiation in MSCs by showing the relation between their expression and some key genes involved in osteoblastic differentiation. In addition, we found a connection between the Twist1 expression level and osteoblastic differentiation by using a Twist‐inhibitor (metformin). - Journal of Cellular Physiology, EarlyView.