Severe radiation-induced skin injury is a complication of tumor radiotherapy and nuclear accidents. Cell therapy is a potential treatment for radiation-induced skin injury. The stromal vascular fraction (SVF) is a newer material in stem cell therapy that is made up of stem cells harvested from adipose tissue, which has been shown to promote the healing of refractory wounds of different causes. In this study, SVF was isolated from patients with radiation-induced skin injury. Adipose-derived stem cells (ADSCs) accounted for approximately 10% of the SVF by flow cytometry. Compared with the control group of rats, administration with SVF attenuated the skin injury induced by electron beam radiation. The effect of SVF on the human skin fibroblast microenvironment was determined by proteomic profiling of secreted proteins in SVF-co-cultured human skin fibroblast WS1 cells. Results revealed 293 upregulated and 1,481 downregulated proteins in the supernatant of SVF-co-cultured WS1 cells. WS1 co-culture with SVF induced secretion of multiple proteins including collagen and MMP-1. In the clinic, five patients with radiation-induced skin injury were recruited to receive SVF transfer-based therapy, either alone or combined with flap transplantation. Autogenous SVF was isolated and introduced into a multi-needle precision electronic injection device, which automatically and aseptically distributed the SVF to the exact layer of the wound in an accurate amount. After SVF transfer, wound healing clearly improved and pain was significantly relieved. The patients' skin showed satisfactory texture and shape with no further wound recurrence. Our findings suggest that transplantation of SVF could be an effective countermeasure against severe radiation-induced skin injury.