The modern diet delivers nearly equal amounts of carbohydrates and protein into the colon representing an important protein increase compared to past higher fiber diets. At the same time, plant-based protein foods have become increasingly popular, and these sources of protein are generally less digestible than animal protein sources. As a result, a significant amount of protein is expected to reach the colon and be available for fermentation by gut microbiota. While studies on diet-microbiota interventions have mainly focused on carbohydrate fermentation, limited attention has been given to the role of protein or protein-fiber mixtures as fermentation substrates for the colonic microbiota. In this study, we aimed to investigate: (1) how changing the ratio of protein to fiber substrates affects the types and quantities of gut microbial metabolites and bacteria; and (2) how the specific fermentation characteristics of different types of fiber might influence the utilization of protein by gut microbes to produce beneficial short chain fatty acids. Our results revealed that protein fermentation in the gut plays a crucial role in shaping the overall composition of microbiota communities and their metabolic outputs. Surprisingly, butyrate production was maintained or increased when fiber and protein were combined, and even when pure protein samples were used as substrates. These findings suggest that indigestible protein in fiber-rich substrates may promote the production of microbial butyrate perhaps including the later stages of fermentation in the large intestine.