Radiation-induced muscle fibrosis is a long-term side effect of radiotherapy that significantly affects the quality of life and even reduces the survival of cancer patients. We have demonstrated that radiation induces satellite cell (SC) activation at the molecular level; however, cellular evidence in a rat model of radiation-induced muscle fibrosis was lacking. In this study, we evaluated SC activation in vivo and investigated whether radiation affects the proliferation and differentiation potential of SCs in vitro. For in vivo studies, Sprague-Dawley rats were randomly divided into six groups (n = 6 per group): non-irradiated controls, 90 Gy/1 week-, 90 Gy/2 weeks-, 90 Gy/4 weeks-, 90 Gy/12 weeks- and 90 Gy/24 weeks-postirradiation groups. Rats received a single dose of radiation in the left groin area and rectus femoris tissues were collected in the indicated weeks. Fibrosis, apoptosis, and autophagy were evaluated by Masson's trichrome staining, TUNEL staining, and electron microscopy, respectively. SC activation and central nuclear muscle fibers were evaluated by immunofluorescence staining and hematoxylin and eosin staining. IL-1β concentrations in serum and irradiated muscle tissue samples were determined by ELISA. For in vitro studies, SCs were isolated from rats with radiation-induced muscle fibrosis and their proliferation and differentiation were evaluated by immunofluorescence staining. In vivo, fibrosis increased over time postirradiation. Apoptosis and autophagy levels, IL-1β concentrations in serum and irradiated skin tissues, and the numbers of SCs and central nuclear muscle fibers were increased in the irradiated groups when compared with the control group. In vitro, cultured SCs from irradiated muscle were positive for the proliferation marker Pax7, and differentiated SCs were positive for the myogenic differentiation marker MyHC. This study provided cellular evidence of SC activation and proliferation in rats with radiation-induced muscle fibrosis.