Background Chronic alcohol exposure impairs both the systemic and intestinal immune system. In humans, alcohol use disorder increases sepsis mortality. However, the mechanisms by which alcohol impacts immune dysregulation during sepsis are not well understood. We previously showed that alcohol downregulates the integrin CD103 on regulatory T cells (Tregs), a highly specialized immunosuppressive T cell population, in non-septic mice. We hypothesized that alcohol-induced changes in CD103 signaling on Tregs would increase sepsis mortality. Methods To model chronic alcohol ingestion, B6 mice were randomized to drink either water or alcohol for 12 weeks. At 18-22 weeks, mice underwent cecal-ligation-and-puncture (CLP) to induce polymicrobial intraabdominal sepsis. Mice were sacrificed after 24 hours, and single cell suspensions were prepared from the spleen, lungs, and small intestine for flow cytometric analysis. Comparisons between groups were analyzed using Student’s T Test or Mann Whitney Test. For survival analysis, sex-matched B6 mice underwent CLP. Mice were randomized to receive injections of CD103 blocking antibody or control postoperatively. For Treg depletion, mice received injections of anti-CD25 prior to and at the time of CLP. Mice were followed for seven days, and their survival was compared using Kaplan-Meier curves. Results Frequencies of T cell populations in the spleen, lung, and intestine of septic water-fed versus alcohol-fed mice were assessed by flow cytometry. Compared to water-fed mice, alcohol-fed mice had lower frequencies of CD103+Foxp3+CD4+Tregs in the spleen (p=0.0004), lungs (p=0.008), small intestine epithelium (p=0.0004) and lamina propria (p=0.0002). Compared to CD103- Tregs, CD103+ Tregs had higher expression of activation markers (CD44, CD69) and immunosuppressive molecules (CTLA4, PD1, CD39, CD73, TIGI), whereas CD103- Tregs expressed higher levels of a naïve circulating marker (CD62L) and transcription factor associated with the pro-inflammatory Th17 phenotype (RORyt). Tregs from the epithelium of septic alcohol-fed mice expressed higher levels of CD44 (p=0.02) and RORyt (p=0.04) than those from water-fed mice. CD103 blockade significantly increased sepsis mortality in water-fed mice (p=0.03), but did not affect survival in alcohol-fed or Treg-depleted mice. Conclusion Alcohol causes a shift in the Treg population by decreasing the frequency of the highly immunosuppressive CD103+Treg subset during sepsis and a change in Treg phenotype in the small intestine epithelium. CD103 signaling has a protective effect in sepsis. This effect is mediated, in part, by Tregs and eliminated by chronic alcohol exposure. Alcohol-induced downregulation of CD103 on Tregs therefore contributes to immune dysregulation in sepsis.