The vagus nerve is proposed to enable communication between the gut microbiome and brain, but activity-based evidence is lacking. Herein, we assess the extent of microbial influences on vagal activity and metabolite signaling mechanisms involved. We find that mice reared without microbiota (germ-free, GF) exhibit decreased vagal afferent tone relative to conventionally colonized mice (specific pathogen-free, SPF), which is reversed by colonization with SPF microbiota. Perfusing non-absorbable antibiotics (ABX) into the duodenum of SPF mice, but not GF mice, acutely decreases vagal activity, which is restored upon re-perfusion with bulk lumenal contents or sterile filtrates from the small intestine and cecum of SPF, but not GF, mice. Of several candidates identified by metabolomic profiling, microbiome- and diet-dependent short-chain fatty acids, bile acids, and 3-indoxyl sulfate stimulate vagal activity with varied response kinetics, which is blocked by co-perfusion of pharmacological antagonists of FFAR2, TGR5, and TRPA1, respectively, into the small intestine. At the single-unit level, duodenal serial perfusion of each metabolite class elicits more singly responsive neurons than dually responsive neurons, suggesting distinct neuronal detection of different microbiome- and macronutrient- dependent metabolites. Finally, microbial metabolite-induced increases in vagal activity correspond with activation of neurons in the nucleus of the solitary tract in the corresponding receptor-dependent manner. Results from this study reveal that the gut microbiome regulates select lumenal metabolites that differentially activate chemosensory vagal afferent neurons, thereby enabling microbial signaling across the gut-brain axis.