Altered intestinal microbial composition promotes intestinal barrier dysfunction and triggers the initiation and recurrence of inflammatory bowel disease (IBD). Current treatments for IBD are focused on control of inflammation rather than on maintaining intestinal epithelial barrier function. Here, we show that the internalization of Gram‐negative bacterial outer membrane vesicles (OMVs) in human intestinal epithelial cells promotes recruitment of caspase‐5 and PIKfyve to early endosomal membranes via sorting nexin 10 (SNX10), resulting in LPS release from OMVs into the cytosol. Caspase‐5 activated by cytosolic LPS leads to Lyn phosphorylation, which in turn promotes nuclear translocalization of Snail/Slug, downregulation of E‐cadherin expression, and intestinal barrier dysfunction. SNX10 deletion or treatment with DC‐SX029, a novel SNX10 inhibitor, rescues OMV‐induced intestinal barrier dysfunction and ameliorates colitis in mice by blocking cytosolic LPS release, caspase‐5 activation, and downstream signaling. Our results show that targeting SNX10 may be a new therapeutic approach for restoring intestinal epithelial barrier function and promising strategy for IBD treatment.