Objective:To explore the therapeutic outcome of rat skeletal muscle extracellular matrix(ECM) hydrogels blended with skeletal muscle stem cells for skeletal muscle defects in a rat model.Methods:From May, 2018 to January, 2020, 5 methods were employed to obtain ECM from rat skeletal muscle. The resulting ECM was evaluated using haematoxylin and eosin (HE) staning and 4-6-diamidino-2-phenylindole (DAPI) staining. The components and structure of hydrogel were assessed by Masson staining and scanning electron microscopy (SEM). Skeletal muscle stem cells were then blended in the hydrogel and MTT was used to observe the growth of cells. Rat skeletal muscle defect models were established and HE staining was used to examine the growth of injured skeletal muscle in each group. Expression of Pax7 and MyoD were detected by immunofluorescence in each group. The function of skeletomuscular tissue was evaluated by electrical stimulation in each group. T test and ONE-WAY analysis of variance were used for data analysis.Results:In this study, a method that caused little damage to skeletal muscle structure and completely removed the cell components was found. This method was employed in the preparation of hydrogel. Masson staining and SEM showed that hydrogel retained the basic structure and some biological factors of skeletal muscle. MTT test showed that hydrogel promoted the proliferation of skeletal muscle stem cells. The experiments in rat skeletal muscle defect models showed that the group treated with stem cells [Specific force was (9.00±2.10) N/cm 2 achieved better outcomes than the other groups (4.06±1.12) N/cm 2 and (5.00±1.60) N/cm 2 in unrepaired group and hyclrogel group, respectively] ( P<0.05) . Conclusion:The results indicated that ECM hydrogel has a good biocompatibility. Furthermore, it was observed that rat skeletal muscle ECM hydrogels blended with skeletal muscle stem cells promoted the repair of muscle defect in a rat model, which would offer a promising strategy for functional muscle repair.