Bile acids (BAs) play important roles in lipid homeostasis, and BA signaling pathways serve as therapeutic targets for nonalcoholic fatty liver disease (NAFLD). Recently, we generated cytochrome P450, family 2, subfamily C, polypeptide 70 (Cyp2c70) mice with a human-like BA composition lacking mouse-/rat-specific muricholic acids to accelerate translation from mice to humans. We employed this model to assess the consequences of a human-like BA pool on diet-induced obesity and NAFLD development. Male and female Cyp2c70 mice and WT littermates were challenged with a 12-week high-fat Western-type diet (WTD) supplemented with 0.25% cholesterol. Cyp2c70 deficiency induced a hydrophobic BA pool with high abundances of chenodeoxycholic acid, particularly in females, because of sex-dependent suppression of sterol 12α-hydroxylase (Cyp8b1). Plasma transaminases were elevated, and hepatic fibrosis was present in Cyp2c70 mice, especially in females. Surprisingly, female Cyp2c70 mice were resistant to WTD-induced obesity and hepatic steatosis, whereas male Cyp2c70 mice showed similar adiposity and moderately reduced steatosis compared with WT controls. Both intestinal cholesterol and FA absorption were reduced in Cyp2c70 mice, the latter more strongly in females, despite unaffected biliary BA secretion rates. Intriguingly, the biliary ratio 12α-/non-12α-hydroxylated BAs significantly correlated with FA absorption and hepatic triglyceride content as well as with specific changes in gut microbiome composition. The hydrophobic human-like BA pool in Cyp2c70 mice prevents WTD-induced obesity in female mice and NAFLD development in both genders, primarily because of impaired intestinal fat absorption. Our data point to a key role for 12α-hydroxylated BAs in control of intestinal fat absorption and modulation of gut microbiome composition.