Bile acids (BAs) are surfactant molecules that regulate the intestinal absorption of lipids. Thus, the modulation of BAs represents a potential therapy for nonalcoholic fatty liver disease (NAFLD), which is characterized by hepatic accumulation of fat and is a major cause of liver disease worldwide. Cyp8b1 is a critical modulator of the hydrophobicity index of the BA pool. As a therapeutic proof of concept, we aimed to determine the impact of Cyp8b1 inhibition in vivo on BA pool composition and as protection against NAFLD. Inhibition of Cyp8b1 expression in mice led to a remodeling of the BA pool, which altered its signaling properties and decreased intestinal fat absorption. In a model of cholesterol-induced NAFLD, Cyp8b1 knockdown significantly decreased steatosis and hepatic lipid content, which has been associated with an increase in fecal lipid and BA excretion. Moreover, inhibition of Cyp8b1 not only decreased hepatic lipid accumulation, but also resulted in the clearance of previously accumulated hepatic cholesterol, which led to a regression in hepatic steatosis. Taken together, our data demonstrate that Cyp8b1 inhibition is a viable therapeutic target of crucial interest for metabolic diseases, such as NAFLD. Chevre, R., Trigueros-Motos, L., Castano, D., Chua, T., Corlian, M., Patankar, JV, Sng, L., Sim, L., Juin, TL, Carissimo, G., Ng, LFP, Yi, CNJ, Eliathamby, CC, Groen, AK, Hayden, MR, Singaraja, RR Therapeutic modulation of the bile acid pool by Cyp8b1 knockdown protects against nonalcoholic fatty liver disease in mice.