Previous research has indicated that chenodeoxycholic acid (CDCA) plays a significant role in intestinal motility and is implicated in some gastrointestinal (GI) disorders. However, the impact of CDCA on defecation patterns in infants, particularly those who are breastfed, remains unclear. This study explored the relationship between CDCA and defecation frequency in breastfed infants, focusing on the underlying mechanisms.We measured CDCA levels in stool samples of infants with varying defecation frequencies using metabolomics, along with studies to explore the impact of CDCA on murine colonic transit and with in vitro cellular studies to elucidate the underlying physiological mechanisms.In our murine model, CDCA treatment increased GI transit distance, enhanced fecal water content, and reduced transit time, without causing significant colonic damage. Additionally, CDCA treatment enhanced 5-hydroxytryptamine (5-HT) secretion and upregulated signaling molecules such as tryptophan hydroxylase 1 (Tph1), G protein-coupled bile acid receptor 1 (TGR5), and transient receptor potential ankyrin 1 (TRPA1). Selective inhibition of these pathways suppressed 5-HT secretion and normalized intestinal motility parameters, confirming their mechanistic role. Moreover, CDCA treatment of rat insulinoma-derived RIN-14B cells also resulted in increased 5-HT secretion.These findings reveal that CDCA significantly correlates with defecation frequency in breastfed infants and likely influences intestinal motility through the TGR5/TRPA1-5-HT signaling axis.Copyright © 2025 AME Publishing Company. All rights reserved.