Extracellular HMGB1 (high mobility group box 1) is considered as a damage-associated molecular pattern protein. However, little is known about its intracellular role. We studied the mechanism whereby intestinal epithelial HMGB1 contributes to host defense, using cell culture, colonoids, conditional intestinal epithelial HMGB1-knockout mice with Salmonella-colitis, il10-/- mice, and human samples. We report that intestinal HMGB1 is an important contributor to host protection from inflammation and infection. We identified a physical interaction between HMGB1 and STAT3. Lacking intestinal epithelial HMGB1 led to redistribution of STAT3 and activation of STAT3 post bacterial infection. Indeed, Salmonella-infected HMGB1-deficient cells exhibited less macroautophagy/autophagy due to decreased expression of autophagy proteins and transcriptional repression by activated STAT3. Then, increased p-STAT3 and extranuclear STAT3 reduced autophagic responses and increased inflammation. STAT3 inhibition restored autophagic responses and reduced bacterial invasion in vitro and in vivo. Moreover, low level of HMGB1 was correlated with reduced nuclear STAT3 and enhanced p-STAT3 in inflamed intestine of il10-/- mice and inflammatory bowel disease (IBD) patients. We revealed that colonic epithelial HMGB1 was directly involved in the suppression of STAT3 activation and the protection of intestine from bacterial infection and injury. Abbreviations: ATG16L1: autophagy-related 16-like 1 (S. cerevisiae); DAMP: damage-associated molecular pattern; HBSS: Hanks balanced salt solution; HMGB1: high mobility group box 1; IBD: inflammatory bowel disease; IL1B/Il-1β: interleukin 1 beta; IL10: interleukin 10; IL17/IL-17: interleukin 17; MEFs: mouse embryonic fibroblasts; STAT3: signal transducer and activator of transcription 3; TLR: toll-like receptor; TNF/TNF-α: tumor necrosis factor.