R-Spondin1 (RSpo1) is a novel secreted protein that augments canonical Wnt/ -catenin signaling. We injected recombinant RSpo1 protein into transgenic Wnt reporter TOPGAL mice and have identified the oral mucosa as a target tissue for RSpo1. Administration of RSpo1 into normal mice triggered nuclear translocation of -catenin and resulted in increased basal layer cellularity, thickened mucosa, and elevated epithelial cell proliferation in tongue. We herein evaluated the therapeutic potential of RSpo1 in treating chemotherapy or radiotherapyinduced oral mucositis in several mouse models. Prophylactic treatment with RSpo1 dose-dependently overcame the reduction of basal layer epithelial cellularity, mucosal thickness, and epithelial cell proliferation in tongues of mice exposed to whole-body irradiation. RSpo1 administration also substantially alleviated tongue mucositis in the oral cavity of mice receiving concomitant 5-fluorouracil and x-ray radiation. Furthermore, RSpo1 significantly reduced the extent of tongue ulceration in mice receiving a single fraction, high dose head-only radiation in a dose-dependent manner. Moreover, combined therapy of RSpo1 and keratinocyte growth factor resulted in complete healing of tongue ulcers in mice subjected to snout-only irradiation. In conclusion, our results demonstrate RSpo1 to be a potent therapeutic agent for oral mucositis by enhancing basal layer epithelial regeneration and accelerating mucosal repair through upregulation of Wnt/ -catenin pathway.