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Muscle builders frequently consume protein supplements, but little is known about their effect on the gut microbiota. This study compared the gut microbiome and metabolome of self-identified muscle builders who did or did not report consuming a protein supplement. Twenty-two participants (14 males and 8 females) consumed a protein supplement (PS), and seventeen participants (12 males and 5 females) did not (No PS). Participants provided a fecal sample and completed a 24-h food recall (ASA24). The PS group consumed significantly more protein (118 ± 12 g No PS vs. 169 ± 18 g PS, <i>p =</i> 0.02). Fecal metabolome and microbiome were analyzed by using untargeted metabolomics and 16S rRNA gene sequencing, respectively. Metabolomic analysis identified distinct metabolic profiles driven by allantoin (VIP score = 2.85, PS 2.3-fold higher), a catabolic product of uric acid. High-protein diets contain large quantities of purines, which gut microbes degrade to uric acid and then allantoin. The bacteria order Lactobacillales was higher in the PS group (22.6 ± 49 No PS vs. 136.5 ± 38.1, PS (<i>p =</i> 0.007)), and this bacteria family facilitates purine absorption and uric acid decomposition. Bacterial genes associated with nucleotide metabolism pathways (<i>p <</i> 0.001) were more highly expressed in the No PS group. Both fecal metagenomic and metabolomic analyses revealed that the PS group’s higher protein intake impacted nitrogen metabolism, specifically altering nucleotide degradation.