Vault RNAs (vtRNAs) are small noncoding RNAs and highly expressed in many eukaryotes. Here, we identified vtRNA2‐1 as a novel regulator of the intestinal barrier via interaction with RNA‐binding protein HuR. Intestinal mucosal tissues from patients with inflammatory bowel diseases and from mice with colitis or sepsis express increased levels of vtRNAs relative to controls. Ectopically expressed vtRNA2‐1 decreases the levels of intercellular junction (IJ) proteins claudin 1, occludin, and E‐cadherin and causes intestinal epithelial barrier dysfunction in vitro, whereas vtRNA2‐1 silencing promotes barrier function. Increased vtRNA2‐1 also decreases IJs in intestinal organoid, inhibits epithelial renewal, and causes Paneth cell defects ex vivo. Elevating the levels of tissue vtRNA2‐1 in the intestinal mucosa increases the vulnerability of the gut barrier to septic stress in mice. vtRNA2‐1 interacts with HuR and prevents HuR binding to claudin 1 and occludin mRNAs, thus decreasing their translation. These results indicate that vtRNA2‐1 impairs intestinal barrier function by repressing HuR‐facilitated translation of claudin 1 and occludin.