Proteinase-activated receptor 1 (PAR₁)-mediated inflammation remains poorly understood. Here we characterize previously unrecognized effects of PAR₁-induced apoptosis signaling, which contributes to epithelial barrier dysfunction. Incubation of epithelial cells with PAR₁ agonists induced apoptosis and increased epithelial permeability in a caspase-3-dependent manner. Similarly, studies in vivo demonstrated that intracolonic infusion with PAR₁ agonists increased colonic permeability in mice, and that this effect was abolished by pretreatment with a caspase-3 inhibitor. PAR₁ agonists induced tight junctional zonula-occludens 1 disruption and apoptotic nuclear condensation. Investigation into signaling pathways showed that these effects were dependent on caspase-3, tyrosine kinase, and myosin light chain kinase. Conversely, the Src kinase inhibitor PP1 augmented zonula-occludens 1 injury and nuclear condensation induced by PAR₁ agonists. These results support a role for proteinases and PARs in intestinal disease and provide new directions for possible therapeutic applications of PAR₁ antagonists.