Colorectal cancer stem cells (CSCs) contribute to colorectal tumorigenesis and metastasis. Colorectal CSCs reside within specialized niches and harbor self-renewal and differentiation capacities. However, the niche regulations of CSCs remain unclear. Here, we show that intestinal nerve cells are required for CSC self-renewal and colorectal tumorigenesis. Enteric serotonergic neurons produce 5-hydroxytryptamine (5-HT) to function as a modulator of CSC self-renewal. 5-HT receptors HTR1B/1D/1F are highly expressed in colorectal CSCs and engage with 5-HT to initiate Wnt/β-catenin signaling. Mechanistically, colorectal cancer (CRC)-enriched microbiota metabolite isovalerate suppresses the enrichment of the NuRD complex onto Tph2 promoter to initiate Tph2 expression, leading to 5-HT production. 5-HT signaling is correlated with CRC severity. Blocking 5-HT signaling in mice not only inhibits the self-renewal of colorectal CSCs but also displays therapeutic efficacy against CRC tumors. Our findings reveal a cross talk between intestinal neurons and tumor cells that serves as an additional layer for CSC regulation.