Background: Mucositis is a common side effect of cancer therapies and transplant conditioning regimens. Management of mucositis involves multiple approaches from oral hygiene, anti-inflammatory, anti-apoptotic, cytoprotective and antioxidant agents, to cryo-, physical therapy, and growth factors. There is room for novel, affordable treatment options or improvement of currently available therapies. Vitamin D (Vit D) has been shown to regulate mucosa- resident cell populations such as Th17 or innate lymphoid cells and critical mucosal cytokine IL-22, however their therapeutic potential has not been put to test in preclinical mouse models. In this study, we aimed to test the therapeutic potential of Vit D injections and IL-22 overexpression in a murine model of chemotherapy-induced mucositis. Methods: Balb/c mice were given daily intraperitoneal injections of Vit D. Another group received IL-22 plasmid via hydrodynamic gene delivery. Mucositis was induced by methotrexate. Weight loss, intestinal histopathology and IL-22, IL-17A and GM-CSF protein levels in intestinal tissue were measured. Intestinal Il23, Ifng, Tnfa and Il10 gene expression were analyzed by real-time qPCR. Intestinal lamina propria B cell, neutrophil and total innate lymphoid cells were quantified. Results: Daily Vit D injections significantly ameliorated intestinal inflammation and elevated intestinal IL-22 levels compared with control groups. Temporal overexpression of IL-22 by hydrodynamic gene delivery slightly increased intestinal IL-22 but failed to confer significant protection from mucositis. Conclusion: To our knowledge, this is the first experimental demonstration in animal model of mucositis that Vit D and IL-22 supplementation may be beneficial and warrants further trials in human patients.