Tendon-bone interface (TBI) healing in chronic rotator cuff injury (CRCI) in older individuals is a common clinical challenge due to cellular senescence, as well as decreased tissue repair and regeneration. Many studies have demonstrated the anti-aging, improved tissue repair, and bone regeneration properties of rapamycin (RPM) in multiple age-related diseases. This study aimed to explore the effects of RPM on TBI healing after CRCI in an aging rat model.A CRCI model was established in 60 Sprague-Dawley rats (24 months old). Rats were then randomly allocated into the control, 0.1 μg RPM, and 1 μg RPM groups. At 4 and 8 weeks post-reconstructive surgery, the supraspinatus tendon-humerus complexes were harvested for biomechanical, microimaging, histological, and immunohistochemical evaluations.Biomechanical testing results demonstrated that the failure load, ultimate strength, and stiffness of the two RPM groups were significantly higher than those of the control group at 4 and 8 weeks postoperatively. Microradiographically, both RPM groups had significantly higher values of bone mineral density and the ratio of trabecular bone volume to total volume than controls at each time point. Moreover, the RPM groups had higher histological scores and showed better regenerated TBI, characterized by better organizational tissue, more fibrocartilage cells, and more bone formation. Immunohistochemical evaluations showed that RUNX2-, SOX9-, and SCX-positive cells were significantly more in the two RPM groups than in the controls at each time point.RPM may effectively enhance CRCI healing after reconstruction by facilitating osteogenesis, tenogenesis, and fibrocartilage reformation at the TBI, as well as improving biomechanical properties.Copyright © 2024. Published by Elsevier Inc.