The intestinal mucus layer is a highly dynamic interface that protects the intestinal epithelium and underlying tissues against noxious stimuli in the external environment. Alterations to the mucus matrix or the mucus-secreting goblet cells can result in impaired barrier function, lending to translocation of luminal antigens and opportunistic pathogens. Giardia duodenalis - a cosmopolitan parasite that colonizes the small intestine – is one of the leading causes of infectious diarrhea and a major contributor to post-infectious functional gastrointestinal disorders. This study reveals a complex interaction at the surface of intestinal epithelial cells, between G. duodenalis and the intestinal mucus layer. Here, we reveal mechanisms whereby G. duodenalis evades and disrupts the first line of host defense, the mucus layer, by degrading mucin-2 (MUC2), depleting mucin stores, and inducing differential gene expression in the small and large intestine. Infection in Muc2-/- mice demonstrated the importance of a functional mucus layer, as mice devoid of intestinal mucus had elevated trophozoite colonization in the small intestine and suffered significant weight loss. Wild-type mice infected with G. duodenalis had a thinner mucus layer and demonstrated differential mucin gene expression, including an increase in Muc2 and de novo expression of Muc5ac in the colon. Human colonic biopsies exposed to G. duodenalis exhibited a reduction of mucus stores. In vitro, G. duodenalis depleted mucins from human LS174T goblet cells and induced elevated expression of MUC2 mRNA. Mucin depletion was dependent on protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) cell-signaling. Overall, the effects of G. duodenalis on the intestinal mucus barrier resulted in impaired barrier function, observed by the translocation of bacterial species into the extra-intestinal organs of infected mice. Importantly, the broad-spectrum cysteine protease inhibitor E64 prevented mucus degradation, mucin depletion, and the increase in MUC2 gene expression. This study demonstrates a critical role for Giardia’s cysteine proteases in pathogenesis, and describes how Giardia’s disruptions of the mucus barrier facilitate bacterial translocation that may contribute to the onset and propagation of disease.