Gastrointestinal ulcers and bleeding are serious complications of non-steroidal anti-inflammatory drug (NSAID) use. While administration of antibiotics and Toll-like receptor 4 knockdown mitigate NSAID-induced enteropathy, the molecular mechanism of these effects is poorly understood. Intestinal hyperpermeability is speculated to trigger the initial damage due to NSAID use. Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel expressed throughout the gastrointestinal (GI) tract epithelium, which is activated by temperature, extension, and chemicals such as 5,6-epoxyeicosatrienoic acid (5,6-EET). The aim of this study was to investigate the possible role of TRPV4 in NSAID-induced intestinal damage. TRPV4 mRNA and protein expression were confirmed by RT-PCR and immunochemistry, respectively, in mouse and human tissues while TRPV4 channel activity of the intestinal cell line IEC-6 was assessed by Ca²⁺-imaging analysis. TRPV4 activators or the NSAID indomethacin significantly decreased transepithelial resistance (TER) in IEC-6 cells, and indomethacin-induced TER decreases were inhibited by specific TRPV4 inhibitors or siRNA TRPV4 knockdown, as well as by the epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH), which decreased 5,6-EET levels. In TRPV4 knockout mice (TRPV4KO), indomethacin-induced intestinal damage was significantly reduced compared to WT mice. Taken together, these results show that TRPV4 activation in the intestinal epithelium caused epithelial hyperpermeability in response to NSAID-induced arachidonic acid metabolites and contributed to NSAID-induced intestinal damage. Thus, TRPV4 could be a promising new therapeutic target for the prevention of NSAID-induced intestinal damage.