While muscle and bone adaptations to deconditioning have been widely described, few studies have focused on the tendon enthesis. Our study examined the effects of mechanical loading on the structure and mechanical properties of the Achilles tendon enthesis. We assessed the fibrocartilage surface area, the organization of collagen, the expression of collagen II, the presence of osteoclasts, and the tensile properties of the mouse enthesis both after 14 days of hindlimb suspension (HU) and after a subsequent 6 days of reloading. Although soleus atrophy was severe after HU, calcified fibrocartilage (CFc) was a little affected. In contrast, we observed a decrease in non-calcified fibrocartilage (UFc) surface area, collagen fiber disorganization, modification of morphological characteristics of the fibrocartilage cells, and altered collagen II distribution. Compared to the control group, restoring normal loads increased both UFc surface area and expression of collagen II, and led to a crimp pattern in collagen. Reloading induced an increase in CFc surface area, probably due to the mineralization front advancing toward the tendon. Functionally, unloading resulted in decreased enthesis stiffness and a shift in site of failure from the osteochondral interface to the bone, whereas 6 days of reloading restored the original elastic properties and site of failure. In the context of spaceflight, our results suggest that care must be taken when performing countermeasure exercises both during missions and during the return to Earth.