The intestinal mucus layer is a physical barrier separating the tremendous number of gut bacteria from the host epithelium. Defects in the mucus layer have been linked to metabolic diseases, but previous studies predominantly investigated mucus function during high-caloric/low-fiber dietary interventions, thus making it difficult to separate effects mediated directly through diet quality from potential obesity-dependent effects. As such, we decided to examine mucus function in mouse models with metabolic disease to distinguish these factors. Here we show that, in contrast to their lean littermates, genetically obese (ob/ob) mice have a defective inner colonic mucus layer that is characterized by increased penetrability and a reduced mucus growth rate. Exploiting the coprophagic behavior of mice, we next co-housed ob/ob and lean mice to investigate if the gut microbiota contributed to these phenotypes. Co-housing rescued the defect of the mucus growth rate, whereas mucus penetrability displayed an intermediate phenotype in both mouse groups. Of note, non-obese diabetic mice with high blood glucose levels displayed a healthy colonic mucus barrier, indicating that the mucus defect is obesity- rather than glucose-mediated. Thus, our data suggest that the gut microbiota community of obesity-prone mice may regulate obesity-associated defects in the colonic mucosal barrier, even in the presence of dietary fiber.