The gut microbiota plays a critical role in regulating host serotonin (5-HT) biosynthesis. Indigenous spore-forming bacteria (Sp) from the mouse and human microbiota promote 5-HT biosynthesis from colonic enterochromaffin cells (ECs), which supply 5-HT to the mucosa, lumen, and circulating platelets. Microbiota-dependent effects on gut 5-HT significantly impact host physiology, modulating gastrointestinal (GI) motility and platelet function. Select fecal metabolites, increased by Sp, elevate 5-HT in chromaffin cell cultures, suggesting direct microbial signaling to ECs. Elevating luminal concentrations of microbial metabolites increases colonic and blood 5-HT in germ-free mice. These findings demonstrate that Sp are important modulators of host 5-HT, highlighting the role of host-microbiota interactions in regulating 5-HT-related biological processes. The microbiota promotes 5-HT biosynthesis by elevating TPH1 expression in colonic ECs. Spore-forming microbes from the SPF mouse microbiota (Sp) restore serum and colon 5-HT levels in germ-free mice, indicating their role in promoting 5-HT biosynthesis. Sp colonization increases 5-HT staining in ECs, elevates TPH1 expression, and decreases SLC6A4 expression. Microbial metabolites, such as deoxycholate and α-tocopherol, mediate the effects of the microbiota on host 5-HT. These metabolites promote 5-HT biosynthesis by signaling directly to ECs. The microbiota modulates GI motility and platelet function by affecting 5-HT levels. The findings reveal that indigenous spore-forming microbes promote 5-HT biosynthesis from colonic ECs, modulating 5-HT concentrations in both colon and blood. Select microbial metabolites confer the serotonergic effects of indigenous spore-forming microbes by signaling directly to colonic ECs to promote TPH1 expression and 5-HT biosynthesis. The study highlights the importance of the gut microbiota in regulating host 5-HT levels and its impact on physiological processes such as GI motility and platelet function.The gut microbiota plays a critical role in regulating host serotonin (5-HT) biosynthesis. Indigenous spore-forming bacteria (Sp) from the mouse and human microbiota promote 5-HT biosynthesis from colonic enterochromaffin cells (ECs), which supply 5-HT to the mucosa, lumen, and circulating platelets. Microbiota-dependent effects on gut 5-HT significantly impact host physiology, modulating gastrointestinal (GI) motility and platelet function. Select fecal metabolites, increased by Sp, elevate 5-HT in chromaffin cell cultures, suggesting direct microbial signaling to ECs. Elevating luminal concentrations of microbial metabolites increases colonic and blood 5-HT in germ-free mice. These findings demonstrate that Sp are important modulators of host 5-HT, highlighting the role of host-microbiota interactions in regulating 5-HT-related biological processes. The microbiota promotes 5-HT biosynthesis by elevating TPH1 expression in colonic ECs. Spore-forming microbes from the SPF mouse microbiota (Sp) restore serum and colon 5-HT levels in germ-free mice, indicating their role in promoting 5-HT biosynthesis. Sp colonization increases 5-HT staining in ECs, elevates TPH1 expression, and decreases SLC6A4 expression. Microbial metabolites, such as deoxycholate and α-tocopherol, mediate the effects of the microbiota on host 5-HT. These metabolites promote 5-HT biosynthesis by signaling directly to ECs. The microbiota modulates GI motility and platelet function by affecting 5-HT levels. The findings reveal that indigenous spore-forming microbes promote 5-HT biosynthesis from colonic ECs, modulating 5-HT concentrations in both colon and blood. Select microbial metabolites confer the serotonergic effects of indigenous spore-forming microbes by signaling directly to colonic ECs to promote TPH1 expression and 5-HT biosynthesis. The study highlights the importance of the gut microbiota in regulating host 5-HT levels and its impact on physiological processes such as GI motility and platelet function.