The human intestinal tract is highly colonized by a vast number of microorganisms. Despite this permanent challenge, infections remain rare, due to a very effective barrier defense system. Essential effectors of this system are antimicrobial peptides and proteins (AMPs), which are secreted by intestinal epithelial and lymphoid cells, balance the gut microbial community, and prevent the translocation of microorganisms. Several antimicrobial proteins have already been identified in the gut. Nonetheless, we hypothesized that additional AMPs are yet to be discovered in this setting. Using biological screening based on antimicrobial function, here we identified competent antibacterial activity of high-mobility-group box 2 (HMGB2) against Escherichia coli. By recombinant expression, we confirmed this biologically new antimicrobial activity against different commensal and pathogenic bacteria. In addition, we demonstrated that the two DNA-binding domains (HMG boxes A and B) are crucial for the antibiotic function. We detected HMGB2 in several gastrointestinal tissues by mRNA analysis and immunohistochemical staining. In addition to the nuclei, we also observed HMGB2 in the cytoplasm of intestinal epithelial cells. Furthermore, HMGB2 was detectable in vitro in the supernatants of two different cell types, supporting an extracellular function. HMGB2 expression was not changed in inflammatory bowel disease but was detected in certain stool samples of patients, whereas it was absent from control individuals. Taken together, we characterized HMGB2 as an antimicrobial protein in intestinal tissue, complementing the diverse repertoire of gut mucosal defense molecules.