The environmental risks of prothioconazole (PTC), a popular agricultural fungicide, and its main metabolite, prothioconazole-desthio (PTCd), have attracted more and more attention recently. In this study, the adverse effects of PTC and PTCd on liver function in mice and their underlying mechanisms have been systematically studied from the perspective of gut microbiota. Combining the results of physiological, biochemical, and histopathological analysis showed that PTC and PTCd exposure could cause lipid accumulation and inflammation in the liver of mice. In addition, exposure to PTC and PTCd could also significantly affect the transcriptome of liver tissue, leading to disorders of lipid metabolism of the liver. Particularly, the abundances of bacteria in liver tissues were significantly increased with PTC and PTCd exposure. Further results show that PTC and PTCd could affect the expression of genes related to inflammation and the barrier function in colon tissue, leading to intestinal dysfunction in mice. Last but not least, the results based on 16S rRNA gene sequencing and 1H NMR metabolomics analysis showed that exposure to PTC and PTCd could cause gut microbiota imbalances and cecal content metabolic profile disorders. In short, this study found that PTC and PTCd exposure could cause liver damage in mice by changing the gut microbiota, disrupting the intestinal barrier function and promoting bacterial translocation. These results clarified the key role of gut microbiota in liver damage induced by PTC and PTCd in mice and proposed a new insight into the mechanisms of liver toxicity induced by pesticides through the dialogue of the gut-liver axis.