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Rotator cuff tear (RCT) is among the most common shoulder injuries and is prone to rerupture after surgery. Selecting suitable subpopulations of stem cells as a new specific cell type of mesenchymal stem cells has been increasingly used as a potential therapeutic tool in regenerative medicine. In this study, murine adipose-derived SSEA-4+CD90+PDGFRA+ subpopulation cells were successfully sorted, extracted, and identified. These cells showed good proliferation and differentiation potential, especially in the direction of tendon differentiation, as evidenced by qRT-PCR and immunofluorescence. Subsequently, we established a murine rotator cuff injury model and repaired it with subpopulation cells. Our results showed that the subpopulation cells embedded in a fibrin sealant significantly improved the histological score, as well as the biomechanical strength of the repaired tendon enthesis at four weeks after surgery, compared with the other groups. Hence, these findings indicated that the subpopulation of cells could augment the repaired enthesis and lead to better outcomes, thereby reducing the retear rate after rotator cuff repair. Our study provides a potential therapeutic strategy for rotator cuff healing in the future.