Extracellular matrix (ECM) stiffness plays a crucial role in regulating the osteogenic differentiation of mesenchymal stem cells (MSCs) by modulating energy metabolism. This study demonstrates that stiff ECM promotes glycolysis, oxidative phosphorylation (OXPHOS), and enhances the antioxidant defense system during osteogenic differentiation. Stiff ECM increases mitochondrial fusion by upregulating mitofusin 1 and 2 (MFN1 and MFN2) and inhibiting dynamin-related protein 1 (DRP1), which contributes to osteogenesis. Yes-associated protein (YAP) is a key mechanotransducer that integrates ECM mechanical cues with energy metabolic signaling to regulate MSC fate. YAP impacts glycolysis, glutamine metabolism, mitochondrial dynamics, and mitochondrial biosynthesis, which are essential for stiffness-mediated osteogenic differentiation. Additionally, glycolysis regulates YAP activity through cytoskeletal tension-mediated nuclear deformation. The findings suggest that YAP is an important mechanotransducer that links ECM mechanical cues and energy metabolic signaling to affect MSC fate. This study provides valuable insights for improving scaffold design in bone tissue engineering. The results highlight that engineered ECM properties can regulate YAP-mediated energy metabolism to enhance MSC osteogenesis, offering new strategies for biomaterials in regenerative medicine.Extracellular matrix (ECM) stiffness plays a crucial role in regulating the osteogenic differentiation of mesenchymal stem cells (MSCs) by modulating energy metabolism. This study demonstrates that stiff ECM promotes glycolysis, oxidative phosphorylation (OXPHOS), and enhances the antioxidant defense system during osteogenic differentiation. Stiff ECM increases mitochondrial fusion by upregulating mitofusin 1 and 2 (MFN1 and MFN2) and inhibiting dynamin-related protein 1 (DRP1), which contributes to osteogenesis. Yes-associated protein (YAP) is a key mechanotransducer that integrates ECM mechanical cues with energy metabolic signaling to regulate MSC fate. YAP impacts glycolysis, glutamine metabolism, mitochondrial dynamics, and mitochondrial biosynthesis, which are essential for stiffness-mediated osteogenic differentiation. Additionally, glycolysis regulates YAP activity through cytoskeletal tension-mediated nuclear deformation. The findings suggest that YAP is an important mechanotransducer that links ECM mechanical cues and energy metabolic signaling to affect MSC fate. This study provides valuable insights for improving scaffold design in bone tissue engineering. The results highlight that engineered ECM properties can regulate YAP-mediated energy metabolism to enhance MSC osteogenesis, offering new strategies for biomaterials in regenerative medicine.