The triazole fungicide prothioconazole (PTC) can cause adverse effects in animals, and its main metabolite prothioconazole-desthio (PTC-d) is even much more harmful. 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 total bile acids (TBAs) levels in the serum, liver, and feces. Correspondingly, the transcription of genes involved in bile acids (BAs) disposition was significantly influenced by PTC or PTC-d exposure. Furthermore, the BAs composition of serum BAs was analyzed by LC-MS, and the results indicated that PTC and PTC-d exposure changed the BAs composition, lowered 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, increasing the relative level of Lactobacillus with bile salt hydrolase (BSH) activity. Furthermore, PTC and PTC-d exposure 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.