Background <p>Irisin, a cleavage peptide by fibronectin type III domain-containing protein 5 (FNDC5), has been recognized as a promotor of osteogenesis to alleviate osteoporosis (OP). However, the detailed regulatory mechanism of osteogenesis remains unclear. This study uncovered the mechanisms underlying bone formation.</p> Methods <p>Osteogenic activity was evaluated by ALP and ARS staining. Expression of target molecules and osteogenic-related factors was assessed by RT-qPCR, Western blotting, and immunohistochemical staining. The binding of euchromatic histone lysine methyltransferase 2 (EHMT2) to distal-less homeobox 3 (DLX3) promoter was validated by ChIP and luciferase reporter assays. Protein interaction, ubiquitination, and small ubiquitin-like modifier (SUMO) modification were determined by Co-IP or Ni²⁺-NTA pull-down assay. In vivo bone formation was evaluated in a mouse model. Histological analysis was performed by HE and Masson staining.</p> Results <p>EHMT2 expression was decreased, while DLX3 expression was increased during osteogenic differentiation in vitro. Down-regulation of EHMT2 facilitated osteogenesis via enhancing DLX3 transcription and expression with assistance of H3K9me2. Moreover, EHMT2 was down-regulated by S-phase kinase-associated protein 2 (SKP2)-mediated ubiquitination, whereas protein inhibitor of activated STAT 4 (PIAS4)-mediated SUMOylation exerted an opposite role. Finally, irisin down-regulated EHMT2 via repressing SUMOylation and promoting ubiquitination of EHMT2, thus contributing to bone formation.</p> Conclusion <p>Epigenetic modification-mediated down-regulation of EHMT2 by irisin leads to transcriptional activation of DLX3, thus promoting bone formation.</p>