ABSTRACT Enteric α-defensins, termed cryptdins (Crps) in mice, and lysozymes secreted by Paneth cells contribute to innate host defense in the ileum. Antimicrobial factors, including lysozymes and β-defensins, are often embedded in luminal glycosylated colonic Muc2 mucin secreted by goblet cells that form the protective mucus layer critical for gut homeostasis and pathogen invasion. In this study, we investigated ileal innate immunity against Entamoeba histolytica , the causative agent of intestinal amebiasis, by inoculating parasites in closed ileal loops in Muc2 ^+/+ and Muc2 ^−/− littermates and quantifying Paneth cell localization (lysozyme expression) and function (Crp secretion). Relative to Muc2 ^+/+ littermates, Muc2 ^−/− littermates showed a disorganized mislocalization of Paneth cells that was diffusely distributed, with elevated lysozyme secretion in the crypts and on villi in response to E. histolytica . Inhibition of E. histolytica Gal/GalNAc lectin (Gal-lectin) binding with exogenous galactose and Entamoeba histolytica cysteine proteinase 5 ( Eh CP5)-negative E. histolytica had no effect on parasite-induced erratic Paneth cell lysozyme synthesis. Although the basal ileal expression of Crp genes was unaffected in Muc2 ^−/− mice in response to E. histolytica , there was a robust release of proinflammatory cytokines and Crp peptide secretions in luminal exudates that was also present in the colon. Interestingly, E. histolytica -secreted cysteine proteinases cleaved the proregion of Crp4 but not the active form. These findings define Muc2 mucin as an essential component of ileal barrier function that regulates the localization and function of Paneth cells critical for host defense against microbes.