In the gastrointestinal tract, proteases regulate motility, immunity, mucus cleavage, wound healing and tissue remodeling. A proteolytic imbalance has been described in inflammatory bowel disease, irritable bowel syndrome and celiac disease, but it is unclear if this imbalance is an initiator or consequence of disease. Most studies focus on host-derived proteases, while the role of bacterial proteases are poorly understood. We investigated the effects induced by microbial derived proteases on host immunity and physiology, after gnotobiotic colonization of mice with bacterial strains with either high or low proteolytic activity. Bacterial strains for colonization were isolated from two symptomatic patients with ulcerative colitis (UC). Strains with high proteolytic activity (HPA) or low-proteolytic activity (LPA) were selected based on in vitro proteolytic screening of the individual strains to generate two communities. The HPA community consisted of Clostridium perfringens METW, Pseudomonas aeruginosa C4, Enteroccocus faecalis FAAJ, and Bacteroides fragilis BHI A, and the LPA community was comprised of Escherichia coli K2 aer., Ruminococcus gnavus D5FAA1, Enterococcus faecium CNAG, and Streptococcus salivarius CAN K2. Adult germ-free C57BL/6 mice were colonized with the HPA or LPA community for three weeks, after which fecal proteolytic activity and the effects on host barrier function and immunity were determined. Mice colonized with the HPA had higher overall proteolytic activity, elastase activity, and gelatinase activity in fecal samples than LPA-colonized mice (p<0.001). Increased serum LPS was observed in HPA-colonized mice compared to LPA-colonized mice (p<0.05), and more HPA-colonized mice had translocation of live bacteria to the spleen (p=0.07), but no differences in colonic 51Cr-EDTA flux were detected. Using nanostring technology, we probed the colonic expression of various immune and barrier related genes. HPA-colonized mice had upregulated Lyz1, Hif1a, Cdh1, Tjp1, and Ttf3 expression compared to LPA-colonized mice (p<0.05). In addition, higher CD11b (p<0.01), CCR2, and IL-22ra2 (p<0.05) was detected in HPA-colonized mice. Quantification of polymorphonuclear cells in colonic H&E sections revealed greater inflammatory infiltrate in HPA-colonized mice (p<0.001). Finally, levels of β-defensin were increased in the feces of HPA-colonized, as measured by ELISA (p<0.05). We successfully established an in vivo mouse model of microbial proteolytic imbalance. Colonization with a high proteolytic microbiota induces low-grade inflammation and an altered barrier that may contribute to the onset of gut inflammatory disorders. CCC