Background: The study aimed to evaluate the efficacy and safety of gingival mesenchymal stem cells (GMSCs) from human fetal gingival tissue used for treating gingival defects in a rat model. Methods: GMSCs were isolated from human fetal gingival tissue and identified by flow cytometry for nestin, Oct4, vimentin, NANOG, CD105, and CD90. The immunogenicity of GMSCs was analyzed by mixed lymphocyte reactions; the tumorigenicity of GMSCs was evaluated by xenotransplanting into nude mice. The gingival defect animal model was established by mechanical resection in rats. GMSCs were transplanted into the defective area, and the regeneration of gingival tissue was observed twice weekly. Four weeks after transplantation, the gingival tissue was surgically cut down, and the graft was analyzed by immunohistochemistry staining for human mitochondrial antigens and rat CD3 and CD20. Results: GMSCs from human fetal gingival tissue positively expressed nestin, Oct4, vimentin, NANOG, CD105, and CD90. There was no cell aggregation after mixed lymphocyte reactions, and interleukin-2 did not increase. Inoculation of GMSCs into nude mice for 6 months showed no tumor formation. GMSCs were transplanted into the gingiva defects of rats. One week after transplantation, the defect area was reduced, and after 3 weeks the morphology and color of local gingival tissue was similar to normal gingival tissue, and gingival height was the same as the normal control group. Conclusions: Using GMSCs from human fetal gingival tissue to treat gingival defects is a safe and effective innovative treatment method.