Transient receptor potential vanilloid 6 (TRPV6), which is a highly Ca²⁺-selective ion channel, is expressed in gastrointestinal epithelium and implicated in maintaining Ca²⁺ homeostasis via transcellular Ca²⁺ transport. However, the local pathophysiological roles of TRPV6 in the gastrointestinal tract remains undefined. In the current study, we investigated the role of TRPV6 in the pathogenesis of experimentally-induced colitis in mice. TRPV6-deficient (TRPV6KO) mice were generated by CRISPR-Cas9-mediated genome editing on C57BL/6 background. Experimental colitis was induced in TRPV6KO and wild-type (WT) mice by the treatment with dextran sulfate sodium (DSS) for 7 days. Intestinal permeability was evaluated by FITC-dextran methods. DSS treatment produced body weight loss with diarrhea and blood feces, and severe colitis characterized by shortening colon length and histological injury 7 days later. The severity of colitis with systemic symptoms was significantly augmented in TRPV6KO mice compared with WT mice. Intestinal permeability was increased in TRPV6KO compared with WT mice. The expression of E-cadherin and occludin in the colon was reduced in TRPV6KO mice compared with WT mice. These findings suggest that TRPV6 plays a protective role in the pathogenesis of DSS-induced colitis via maintaining colonic barrier functions. Thus, TRPV6 may be a novel target for the treatment of gastrointestinal diseases.