Cities are among the most extreme forms of anthropogenic ecosystem modification and urbanization processes exert profound effects on animal populations through multiple ecological pathways. Increased access to human associated food items may alter species’ foraging behavior and diet, in turn modifying the normal microbial community of the gastrointestinal tract, ultimately impacting their health. It is crucial we understand the role of dietary niche breadth and the resulting shift in the gut microbiota as urban animals navigate novel dietary resources. We combined stable isotope analysis of hair and microbiome analysis of four gut regions across the gastrointestinal tract to investigate the effects of urbanization on the diet and gut microbiota of two sympatric species of rodent with different dietary niches; the omnivorous large Japanese field mouse (Apodemus speciosus) and the relatively more herbivorous grey red-backed vole (Myodes rufocanus). Both species exhibited an expanded dietary niche width within the urban areas potentially attributable to novel anthropogenic foods and altered resource availability. We detected a dietary shift in which urban A. speciosus consumed more terrestrial animal protein and M. rufocanus more plant leaves and stems. Such changes in resource use may be associated with an altered gut microbial community structure. There was an increased abundance of the presumably probiotic Lactobacillus in the small intestine of urban A. speciosus and potentially pathogenic Helicobacter in the colon of M. rufocanus. Together, these results suggest that even taxonomically similar species may exhibit divergent responses to urbanization with consequences for the gut microbiota and broader ecological interactions.