The neurotransmitter 5-hydroxytryptamine (5-HT), primarily produced by intestinal enterochromaffin (EC) cells, relies on tryptophan hydroxylase 1 (TPH1) for synthesis. Research suggested Bifidobacterium breve CCFM1025’s potential in regulating Tph1 gene expression, maintaining 5-HT levels in stressed mice, but its precise mechanisms were unclear. This study used metabolomic techniques to assess probiotic fermentation products, revealing acetate as the crucial element in Bb-CCFM1025’s regulation of gut 5-HT synthesis. Further exploration correlated acetate with Tph1 transcription in intestinal organoids. Transcriptomic methods and quantitative reverse transcription polymerase chain reaction validation demonstrated how acetate facilitated 5-HT synthesis and secretion. It unveiled that acetate orchestrates signaling pathways (phosphoinositide 3-kinase-protein kinase B (PI3K-AKT), phospholipase C-phosphorylated extracellular signal-regulated kinase (PLC-pERK), and PLC-1,4,5-trisphosphate (IP3)-Ca2+) within EC, enabling 5-HT production. These findings elucidate the biochemical mechanisms behind specific probiotics’ effects, aiding in the targeted selection of similar beneficial strains. This study offers theoretical support for choosing probiotics with analogous functionalities based on their physiological impacts.