Bile acid (BA) metabolism and signaling may influence cancer risk. We investigated differences in circulating BA composition between healthy controls and patients with colorectal cancer (CRC) and assessed the epithelial-mesenchymal transition (EMT) in CRC cell lines exposed to chenodeoxycholic acid (CDCA). BA, C4, and fibroblast growth factor-19 (FGF19) concentrations in serum samples from 70 controls and 133 patients with CRC were analyzed. Results showed that primary and secondary BAs, C4, and FGF19 were significantly lower in CRC patients than in the age- and sex- matched controls (P < 0.05). CDCA reduced the percentage of EdU-positive cells in HCT116 and HT-29 cells by 53.6% and 28.3%, respectively (P < 0.001) and decreased the rate of wound closure by 0.31 ± 0.05- and 0.30 ± 0.06-fold in HCT116 and HT-29 cells, respectively(P < 0.05). It significantly reduced spheroid formation by 90.0% and 94.3% in HCT116 and HT-29 cells, respectively (P < 0.0001) and downregulated EMT-related stem cell markers in CRC cells. The dysregulation of BA synthesis and recycling might contribute to CRC carcinogenesis. CDCA might alter EMT, protecting against CRC development and progression. The predictive role of plasma BAs in CRC prognosis and their potential in CRC prevention and management should be further explored.