Background: Tendons are the force transferring tissue that enable joint movement. Excessive mechanical loading is commonly considered as a primary factor causing tendinopathy, however, an increasing body of evidence supports the hypothesis that overloading creates microdamage of collagen fibers resulting in a localized decreased loading on the cell population within the damaged site. Heterotopic ossification is a complication of late stage tendinopathy, which can significantly affect the mechanical properties and homeostasis of the tendon. Here, we the examine the effect of mechanical underloading on tendon ossification and investigate its underlying molecular mechanism. Method: Rabbit Achilles tendons were dissected and cultured in an underloading environment (3% cyclic tensile stain,0.25 Hz, 8 h/day) for either 10, 15 or 20 days. Using isolated tendon-derived stem cells (TDSCs) 3D constructs were generated, cultured and subjected to an underloading environment for 6 days. Histological assessments were performed to evaluate the structure of the 3D constructs; qPCR and immunohistochemistry were employed to study TDSC differentiation and the β-catenin signal pathway was investigated by Western blotting. Mechanical testing was used to determine ability of the tendon to withstand force generation. Result: Tendons cultured for extended times in an environment of underloading showed progressive heterotopic ossification and a reduction in biomechanical strength. qPCR revealed that 3D TDSCs constructs cultured in an underloading environment exhibited increased expression of several osteogenic genes: these include RUNX2, ALP and osteocalcin in comparison to tenogenic differentiation markers (scleraxis and tenomodulin). Immunohistochemical analysis further confirmed high osteocalcin production in 3D TDSCs constructs subject to underloading. Western blotting of TDSC constructs revealed that β-catenin accumulation and translocation were associated with an increase in phosphorylation at Ser552 and decrease phosphorylation at Ser33. Conclusion: These findings unveil a potential mechanism for heterotopic ossification in tendinopathy due to the underloading of TDSCs at the damage sites, and also that β-catenin could be a potential target for treating heterotopic ossification in tendons. The Translational potential: Tendon heterotopic ossification detrimentally affect quality of life especially for those who has atheletic career. This study reveals the possible mechanism of heterotpic ossification in tendon related to mechanical loading. This study provided the possible to develop a mechanical stimulation protocol for preventive and therapeutic purpose for tendon heterotopic ossification.