BACKGROUND & AIMS: Observational studies have shown that diets high in fat and low in dietary fiber, might have an unfavorable impact on bile acid (BA) profiles, which might further affect host cardiometabolic health. In the current study, we aimed to evaluate the effects of dietary fat content on BA profiles and associated gut microbiota, and their correlates with cardiometabolic risk factors. METHODS: In a randomized controlled-feeding trial, healthy young adults were assigned to one of the three diets: a lower-fat diet (fat 20%, carbohydrate 66% and protein 14%), a moderate-fat diet (fat 30%, carbohydrate 56% and protein 14%) and a higher-fat diet (fat 40%, carbohydrate 46% and protein 14%) for 6 months. All the foods were provided during the entire intervention period. The BA profiles, associated gut microbiota and markers of cardiometabolic risk factors were determined before and after intervention. RESULTS: The higher-fat diet resulted in an elevated concentration of total BAs (p??0.3, p?=?0.02 and p?=?0.008 after FDR correction, respectively). In line with these findings, serum fibroblast growth factor 19 (FGF19) was marginally significantly elevated in the higher-fat group after intervention (p?=?0.05). CONCLUSIONS: The higher-fat diet resulted in an alteration of BAs, especially unconjugated BAs and secondary BAs, most likely through actions of gut microbiota. These alterations might confer potentially unfavorable impacts on colonic and host cardiometabolic health in healthy young adults. Clinical trial registry number: NCT02355795 listed on NIH website: ClinicalTrials.gov.