Cyp2c70knockout mice lack the enzyme that produces muricholic acids and show a “human-like” hydrophobic bile acid pool-induced hepatobiliary injury. In this study, we investigated the potential anti-cholestasis effect of glycine-conjugated β muricholic acid (G-β-MCA) in maleCyp2c70KO mice based on its hydrophilic physiochemical property and signaling property as an farnesoid X receptor (FXR) antagonist. Our results showed that G-β-MCA treatment for 5 weeks alleviated ductular reaction and liver fibrosis and improved gut barrier function. Analysis of bile acid metabolism suggested that exogenously administered G-β-MCA was poorly absorbed in the small intestine and mostly deconjugated in the large intestine and converted to taurine-conjugated MCA (T-MCA) in the liver, leading to T-MCA enrichment in the bile and small intestine. These changes decreased the biliary and intestine bile acid hydrophobicity index. Furthermore, G-β-MCA treatment decreased intestine bile acid absorption via unknown mechanisms, resulting in increased fecal bile acid excretion and a reduction in total bile acid pool size. In conclusion, G-β-MCA treatment reduces the bile acid pool size and hydrophobicity and improves liver fibrosis and gut barrier function inCyp2c70KO mice.