The triazole fungicide prothioconazole (PTC) can cause some adverse effects in animals, and prothioconazole-desthio (PTC-d), as its main metabolite, is even much more harmful than PTC. However, the toxic effects of PTC and PTC-d on the liver-gut axis of mice are still unknown. In the present experiment, we found that oral exposure to PTC and PTC-d increased the relative liver weights and the serum and hepatic total bile acids (TBAs) levels. What’s more, the TBAs levels in the feces also increased. Correspondingly, the transcription of genes connected to hepatic and intestinal bile acids (BAs) metabolism was significantly influenced by PTC or PTC-d exposure. For example, the main genes related to hepatic BAs synthesis increased significantly while the genes related to ileal BAs reabsorption decreased. Furthermore, the BAs composition of serum BAs was analyzed by LC-MS based metabolomic technique, and the results indicated that PTC and PTC-d exposure changed the BAs composition, caused lower the ratio of conjugated/unconjugated BAs, elevated the ratio of CA/b-MCA, and enhanced the hydrophobicity of BAs pool. 16s RNA gene sequencing of the DNA from colonic contents uncovered that PTC and PTC-d exposure altered the relative abundance and constitution of intestinal microbiota, particularly increasing the relative level of Lactobacillus with bile salt hydrolase (BSH) activity. What’s more, PTC and PTC-d exposure not only disrupted the balance of the gut microbiota but also impaired the gut barrier function, causing an increase in mucus secretion. In particular, the effects of PTC-d on some endpoints in the BAs metabolism and gut barrier function had been proven to be more significant than the parent compound PTC. All these findings draw attention to the health risk of PTC and PTC-d exposure in regulating BAs metabolism, which might lead to some metabolic disorders and occur of related diseases in animals.