Background: Cathepsin G (Cat‐G) is a neutrophil serine‐protease found in the colonic lumen of ulcerative colitis (UC) patients. Cat‐G is able to activate protease‐activated receptor‐4 (PAR4) located at the apical side of enterocytes, leading to epithelial barrier disruption. However, the mechanisms through which Cat‐G triggers inflammation are not fully elucidated. The aims of our study were to evaluate in vivo the effects of UC fecal supernatants and Cat‐G on epithelial barrier function and inflammation, and the connection between these two parameters. Methods: Male balb/c mice were used in this study. We evaluated the effect of a 2‐hour intracolonic infusion of 1) fecal supernatants from UC patients pretreated or not with specific Cat‐G inhibitor (SCGI); 2) PAR4‐activating peptide (PAR4‐AP); and 3) Cat‐G on colonic myeloperoxidase (MPO) activity and paracellular permeability (CPP). The involvement of PAR4 was assessed by pretreating animals with pepducin P4pal‐10, which blocks PAR4 signaling. We investigated the role of myosin light chain (MLC) kinase by using its inhibitor, ML‐7, and we determined phosphorylated MLC (pMLC) levels in mice colonic mucosa. Results: UC fecal supernatants, Cat‐G, and PAR4 agonist increased both CPP and MPO activity in comparison with healthy subjects fecal supernatants. ML‐7 inhibited the CPP increase triggered by Cat‐G by 92.3%, and the enhanced MPO activity by 43.8%. Intracolonic infusion of UC fecal supernatant determined an increased phosphorylation level of MLC. Conclusions: These observations support that luminal factors such as Cat‐G play an important proinflammatory role in the pathogenesis of colitis, mainly depending on CPP increase by MLC phosphorylation.