The study investigates the role of gut microbiota in regulating host serotonin (5-HT) levels. The authors demonstrate that indigenous spore-forming bacteria (Sp) from the mouse and human gut microbiota promote 5-HT biosynthesis from colonic enterochromaffin cells (ECs), which supply 5-HT to the mucosa, lumen, and circulating platelets. The microbiota-dependent effects on gut 5-HT significantly impact host physiology, modulating gastrointestinal motility and platelet function. Specific fecal metabolites increased by Sp elevate 5-HT in chromaffin cell cultures, suggesting direct metabolic signaling from gut microbes to ECs. Elevating luminal concentrations of certain microbial metabolites in germ-free mice increases colonic and blood 5-HT. These findings highlight the importance of Sp in modulating host 5-HT and the role of host-microbiota interactions in regulating 5-HT-related biological processes.The study investigates the role of gut microbiota in regulating host serotonin (5-HT) levels. The authors demonstrate that indigenous spore-forming bacteria (Sp) from the mouse and human gut microbiota promote 5-HT biosynthesis from colonic enterochromaffin cells (ECs), which supply 5-HT to the mucosa, lumen, and circulating platelets. The microbiota-dependent effects on gut 5-HT significantly impact host physiology, modulating gastrointestinal motility and platelet function. Specific fecal metabolites increased by Sp elevate 5-HT in chromaffin cell cultures, suggesting direct metabolic signaling from gut microbes to ECs. Elevating luminal concentrations of certain microbial metabolites in germ-free mice increases colonic and blood 5-HT. These findings highlight the importance of Sp in modulating host 5-HT and the role of host-microbiota interactions in regulating 5-HT-related biological processes.