Gut barrier failure has been implicated in the progression from single-organ injury to multiple-organ failure. The unstirred mucus layer is a major component of the physiological gut barrier; its role in acute pancreatitis (AP) is not clearly defined. Rats underwent biliopancreatic duct ligation-induced AP; two controls were used: biliopancreatic duct ligation with drainage and sham duct ligation. After 4.5 h, serum and ascitic amylase activity was measured. Mucus was analyzed for reactive nitrogen intermediate-mediated damage, reactive oxygen species-induced damage, and total antioxidant capacity. Mucus coverage and villous injury were assessed histologically. Ileum permeability was measured by diffusion of a fluorescent Dextran probe. Histology and morphology of the mucus layer were validated in a mouse AP model (intraductal taurocholate plus cerulein). Biliopancreatic duct ligation increased serum α-amylase, ascitic volume, and ascitic α-amylase. Intestinal permeability was increased, which was associated with loss of the unstirred mucus layer but not villous injury. These changes correlated with increased reactive oxygen species- and- reactive nitrogen intermediate-mediated mucus damage as well as decreased mucus total antioxidant capacity but were not present in the two control groups. Using a different model of AP in mice, the finding of mucus layer disruption was recapitulated at 6 h after AP, but by 24 h, rebound hypersecretion of inspissated mucus was seen. These results support the hypothesis that damage to the unstirred mucus layer with evidence of oxidative stress occurs during AP-induced gut barrier failure.