Giardia lamblia is a widespread anaerobic protistan parasite causing significant diarrheal disease worldwide. Giardia trophozoites attach extracellularly to the host gastrointestinal epithelium using a unique microtubule organelle, the ventral disc. The complex, dome-shaped disc is composed of microribbon-crossbridge (MR-CB) protein complexes scaffolded onto a spiral microtubule array. Attachment is dynamic and reversible, facilitating parasite contact and colonization of the gastrointestinal epithelium. To investigate possible contributions of disc-mediated attachment to host pathobiology, we generated a stable quadruple allelic knockout of an abundant disc-associated protein, MBP, using a new method of CRISPR-mediated gene disruption. MBP knockout mutants (MBPKO) had flattened crescent- or horseshoe-shaped discs, severe MR-CB defects, and complete phenotypic penetrance off selection. MBP mutants also had aberrant surface contacts and were unable to resist shear forces under fluid flow. Using a human gastrointestinal organoid model, we discovered that MBPKO mutants had a significantly reduced ability to cause the host epithelial barrier breakdown characteristic of wild-type infections. In contrast, the addition of spent medium or lysed parasites had no impact on epithelial barrier breakdown. Overall, this pioneering work provides direct evidence that MBP is required for the domed disc architecture and that disc-mediated attachment contributes to host pathobiology, specifically epithelial barrier breakdown. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].