Scope: Vitamin D deficiency, which has been a global health issue for decades, is involved in gut health and diseases. We examined the health benefits of Grifola frondosa (maitake mushroom), a potent dietary source of fungal vitamin D (D2), on DSS-induced colitis in vitamin D-deficient mice. Methods and results: C57BL/6J mice were fed a control diet, vitamin D3-deficient diet (DD), maitake-fortified DD, or vitamin D2-fortified DD for 6 weeks. LC-MS/MS analysis demonstrated that maitake-fed mice showed an increased 25(OH)D2 alternative to 25(OH)D3 in plasma, as well as the mice fed an equivalent dose of vitamin D2. The mRNA expression profiles of vitamin D-responsive genes, including renal Cyp24a1 and Cyp27b1, were normalized in the maitake-fed mice. Severe DSS-induced colitis observed in DD mice was attenuated in maitake-fed mice; the accumulation of immune cells in the colonic mucosa and protein expression of colonic claudin-2, a target gene of the vitamin D receptor, were comparable to that in control mice. Conclusion: Dietary intake of maitake was effective in improving vitamin D status and biological function, demonstrating a potential attenuative effect on severe DSS-induced colitis in vitamin D3 deficient mice, as well as equivalent doses of vitamin D2.