Background <p>RNA binding Fox-1 homolog 2 (RBFOX2) is an RNA-binding protein that has been extensively studied in heart disease. Its downstream target, Jph2, has also been primarily investigated in relation to heart disease. However, their roles in osteoblast differentiation remain unexplored. This study aimed to investigate the regulatory role of <i>RBFOX2</i> in osteoblast differentiation through its relationship with <i>Jph2</i> in the MC3T3-E1 preosteoblast cell line.</p> Methods and results <p>The expression levels of <i>RBFOX2</i>, <i>Jph2</i>, and osteoblast differentiation markers (<i>Dlx5</i> and <i>Runx2</i>) were analyzed using RT-PCR, qPCR, and Western blotting. Alkaline phosphatase (ALP) activity and extracellular matrix mineralization were evaluated using ALP and Alizarin Red S staining. Transient overexpression and siRNA-mediated knockdown were performed to assess the functional roles of <i>RBFOX2</i> and <i>Jph2</i> in osteoblast differentiation. Overexpression of RBFOX2 significantly increased Dlx5 and Runx2 expression at both mRNA and protein levels (p &lt; 0.05) and enhanced mineralization in MC3T3-E1 cells. Conversely, knockdown of RBFOX2 or Jph2 resulted in decreased expression of these markers and reduced mineralization. Notably, RBFOX2 was found to upregulate Jph2, and this interaction promoted osteoblast differentiation via modulation of Dlx5 and Runx2.</p> Conclusions <p>These findings suggest that RBFOX2 regulates osteoblast differentiation through Jph2, making it a potential therapeutic target for bone diseases. Further studies are warranted to explore the detailed molecular mechanisms and clinical implications of this regulatory pathway.</p>