The relationship between porcine gut microbiota composition and health is an important area of research, especially due to the need to find alternatives to antimicrobial use to manage disease in livestock production systems. Previous work has indicated that lower crude dietary protein levels can reduce the impacts of post-weaning colibacillosis (PWC), which is a porcine diarrheal disease caused by enterotoxigenic Escherichia coli (ETEC). Here, to explore the complex interactions between the gut microbiota, protein nutrition and ETEC exposure, the microbial compositions of both the ileal digesta and feces were analysed with and without ETEC exposure from pigs fed a low or high protein diet. As ETEC colonisation is mostly localised to the ileum, changes in the small intestinal microbiota were expected in response to ETEC exposure. This was supported by the study findings, which identified significant microbiota changes in ileal samples, but not in fecal samples. Both increased dietary protein and ETEC exposure impacted on ileal microbiota alpha diversity (richness and diversity indices) and beta diversity (structure, stability and relative taxa abundances) at certain sampling points, though the combination of a high protein diet and ETEC exposure had the most profound impact on ileal microbiota composition. An understanding of how infection and nutrition lead to microbiota changes is likely to be required if dietary strategies are to be developed for the management of enteric diseases.