Abstract The understanding of the role of intestinal bacteria in various intestinal diseases has been limited due to the lack of suitable in vitro models. However, recent advancements in Gut-on-a-Chip culture technology provide a promising avenue for exploring complex interactions between intestinal epithelium and bacteria. In this study, we aimed to create a microfluidic Gut-on-a-Chip co-culture system using dogs as a model. Dogs share similarities with humans in terms of intestinal disease pathophysiology, clinical presentations, and intestinal microbiome, making them a relevant model for various intestinal diseases, including inflammatory bowel disease (IBD). We cultured colonoids from both healthy dogs and dogs with IBD on the Gut-on-a-Chip platform to create villus-like structures under dynamic conditions. Co-cultures with non-pathogenic Escherichia coli (NPE) were established in the Gut-on-a-Chip with healthy colonoids, and we conducted a static Transwell (TW) co-culture for comparison. To assess epithelial barrier integrity, we measured transepithelial electrical resistance (TEER), and we evaluated the expression of ZO-1, a tight junction protein, through immunofluorescence staining (IF). Following 6-9 days of uninterrupted medium flow in the Gut-on-a-Chip models, we observed sporadic clusters of villus-like structures (i.e., 3D morphogenesis) in canine intestinal epithelial cells in both the models derived from healthy dogs and those with IBD. The IF confirmed the presence of ZO-1, indicating the establishment of tight junctions. Infection with NPE in the TW system resulted in a significant reduction in TEER (48.2±4.4% at 12 hours and 7.3±5.3% at 24 hours). In contrast, the Gut-on-a-Chip maintained TEER levels at 100.3±12.8% at 12 hours, 94.9±2.2% at 24 hours, and 88.8±15.0% at 48 hours, preserving the 3D structure. The innovative canine Gut-on-a-Chip model, capable of reproducing villus-like structures and preserving a 3D configuration using both healthy and diseased organoids (specifically, IBD), presents a valuable platform for investigating bacterial-epithelium interactions in both human and veterinary medicine. This dynamic microfluidic culture system enables the co-cultivation of living bacteria while maintaining epithelial integrity, facilitating the study of intricate interactions between intestinal bacteria and epithelial crosstalk.