Tendon injuries, commonly associated with sports activities, pose significant challenges in terms of treatment and recovery due to limited tendon regeneration and the formation of proliferative scars. Stem cell-based therapy has shown promising application, but there are still challenges. Physical and biological cues are instrumental in guiding stem cell differentiation and maturation. This study focuses on exploring the effects of matrix biomechanics on tendon stem/progenitor cells (TSPCs) differentiation. We fabricated polydimethylsiloxane (PDMS) substrates with different elastic modulus to mimic the mechanical characteristics of healthy tendons. A tissue-engineered culture system was developed for tenogenesis, and pre-differentiated tissue-engineered tendons were transplanted in vivo to assess their efficacy in regenerating patella tendon injuries. Furthermore, we demonstrated that the biomechanical stimuli activated the integrin-αm to enhance the tenogenesis capacity of TSPCs. Our findings highlight the importance of biomechanics in tendon tissue engineering and provide a novel perspective for enhancing tendon regeneration.