Radiation-induced oral mucositis (RIOM) is one of the most common side effects of radiotherapy in cancer patients, especially in almost all head and neck cancer patients. It presents as severe pain and ulceration. The development of RIOM is composed of five stages: initiation, primary damage response, signal amplification, ulceration, and healing. However, the key regulators involved in the RIOM pathogenesis remain largely unknown. In this study, we reveal a novel role of miR-200c, a member of the miR-200 family, in modulating RIOM pathogenesis. Using a mouse model mimicking RIOM, we found that the miR-200 family numbers (miR-141, miR-200a, miR-200b, and miR-200c) except miR-429 were significantly induced during the RIOM formation. Besides, in RIOM mice, miR-200c expression level was also increased dramatically in the normal human keratinocytes (NHKs) after irradiation. Knockdown of miR-200c expression with miR-200c-3p-shRNA significantly reduced senescence phenotype and enhanced cell proliferation in NHKs after irradiation. The generation of reactive oxygen species (ROS) and p47 enzyme involved in ROS production was increased after irradiation but both were markedly reduced in NHKs by miR-200c inhibition. Knockdown of miR-200c expression in NHKs increased DNA double-strand break repair after irradiation compared with control NHKs. Furthermore, miR-200c inhibition repressed the production of proinflammatory cytokines (TGF-β, TNF-α, and IL-1α) via inhibiting NF-κB and Smad2 activation in NHKs exposed to IR. Additionally, miR-200c inhibition promoted NHK migration and increased the expression of molecules that regulate epithelial to mesenchymal transition, including Snail, Vimentin, Zeb1, and Bmi-1. These results not only identify the key role of miR-200c in the pathogenesis of RIOM but also provide a novel therapeutic target to treat RIOM.