EAEC is a common cause of diarrheal illness worldwide. Pathogenesis is believed to occur in the ileum and colon, where the bacteria adhere and form a robust aggregating biofilm. Among the multiple virulence factors produced by EAEC, the Pic serine protease has been implicated in bacterial colonization by virtue of its mucinolytic activity. Hence, a potential role of Pic in mucus barrier disruption during EAEC infection has been long postulated. In this study, we used human colonoids comprising goblet cells and a thick mucin barrier as an intestinal model to investigate Pic's roles during infection with EAEC. We demonstrated the ability of purified Pic, but not a protease defective Pic mutant to degrade MUC2. Western blot and confocal microscopy analysis revealed degradation of the MUC2 layer in colonoids infected with EAEC, but not with its isogenic EAECpic mutant. Wild-type and MUC2-knockdown colonoids infected with EAEC strains exposed a differential biofilm distribution, greater penetration of the mucus layer and increased colonization of the colonic epithelium by Wild-type EAEC than its isogenic Pic mutant. Higher secretion of pro-inflammatory cytokines was seen in colonoids infected with EAEC than EAECpic. Although commensal E. coli expressing Pic degraded MUC2, it did not show improved mucus layer penetration or colonization of the colonic epithelium. Our study demonstrates a role of Pic in MUC2 barrier disruption in the human intestine and shows that colonoids are a reliable system to study the interaction of pathogens with the mucus layer.