The gut-on-a-chip is a microengineered in vitro model of the living human intestine that reconstitutes the lumen-capillary tissue interface. Intestinal epithelial Caco-2 cells cultured on the extracellular matrix-coated porous membrane in a gut-on-a-chip undergo three-dimensional villus morphogenesis and physiological cytodifferentiation. The gut-on-a-chip closely recapitulates biomechanical and functional characteristics under peristalsis-like cyclic movements and trickling flow, which enables to co-culture gut microbiome with villus epithelium from days to weeks. Host-gut microbiome ecosystem emulated in the gut-on-a-chip allows the pathomimetic modeling of human intestinal diseases such as gut inflammation and bacterial overgrowth. Here, we describe a protocol for microfabrication of a gut-on-a-chip device, reconstitution of intestinal microenvironment, recreation of host-gut microbiome intercellular interactions, and demonstration of the pathophysiology of representative human intestinal diseases associated with the gut microbiome. The modeling of intestinal disease pathophysiology on-chip can potentiate the development of patient-specific disease models that can validate the efficacy and safety of novel therapeutic interventions.