Objective <p>This study investigates how retinoic acid (RA) promotes osteogenic differentiation in mouse facial suture mesenchymal stem cells (MSCs) under mechanical stretching, focusing on the mechanosensitive ion channel <i>Piezo2</i>. We investigate the mechanism underlying the synergistic effects of RA and mechanical stimuli on osteogenic differentiation of MSCs, a process crucial for bone regeneration in craniofacial hypoplasia.</p> Methods <p>Mouse suture MSCs were mechanically stretched with or without RA co-treatment. RNA-seq analyzed gene expression, followed by Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs). Cell stretching and RA-treated murine models were used to validate the findings. Additionally, <i>Piezo2</i>-knockdown MSCs were exposed to mechanical stretching under osteogenic induction conditions to assess depositions of mineralized sites.</p> Results <p>Micro-CT and Immunohistochemistry showed RA-stretch co-treatment significantly widened the zygomaticomaxillary suture (ZMS) compared to controls. This combined treatment induced the expression of osteogenic marker genes. RNA-seq analysis identified <i>Piezo2</i> as a highly upregulated gene (<i>p</i> &lt; 0.0001; Log2(Fold Change) = 4.91), with RT-qPCR validation confirming 8.38-fold higher <i>Piezo2</i> expression in RA-stretch co-treatment groups (<i>p</i> &lt; 0.001). Crucially, <i>Piezo2</i> knockdown prevented the osteogenic differentiation induced by RA and mechanical stretching, evidenced by reduced depositions of mineralized sites.</p> Conclusion <p>These findings indicate that RA promotes osteogenic differentiation of MSCs under mechanical stretching, predominantly through the upregulation of <i>Piezo2.</i> This study provides novel insights into the molecular mechanisms underlying Trans-sutural Distraction Osteogenesis (TSDO) and highlights the potential of RA as an adjunctive therapy for enhancing bone regeneration in craniofacial hypoplasia.</p>