Depletion of gut microbiota is associated with inefficient energy extraction and reduced production of short‐chain fatty acids from dietary fibers, which regulates colonic proglucagon (Gcg) expression and small intestinal transit in mice. However, the mechanism by which the gut microbiota influences dietary protein metabolism and its corresponding effect on the host physiology is poorly understood. Enteropeptidase inhibitors block host protein digestion and reduce body weight gain in diet‐induced obese rats and mice, and therefore they constitute a new class of drugs for targeting metabolic diseases. Enteroendocrine cells (EECs) are dispersed throughout the gut and possess the ability to sense dietary proteins and protein‐derived metabolites. Despite this, it remains unclear if enteropeptidase inhibition affects EECs function. In this study, we fed conventional and antibiotic treated mice a western style diet (WSD) supplemented with an enteropeptidase inhibitor (WSD‐ETPi), analyzed the expression of gut hormones along the length of the intestine, and measured small intestinal transit under different conditions. The ETPi‐supplemented diet promoted higher Gcg expression in the colon and increased circulating Glucagon like peptide‐1 (GLP‐1) levels, but only in the microbiota‐depleted mice. The increase in GLP‐1 levels resulted in slower small intestinal transit, which was subsequently reversed by administration of GLP‐1 receptor antagonist. Interestingly, small intestinal transit was normalized when an amino acid‐derived microbial metabolite, p‐cresol, was supplemented along with WSD‐ETPi diet, primarily attributed to the reduction of colonic Gcg expression. Collectively, our data suggest that microbial dietary protein metabolism plays an important role in host physiology by regulating GLP‐1‐mediated intestinal transit.