OBJECTIVE: A growing body of preclinical and clinical literature suggests that brain-gut-microbiota interactions play an important role in human health and disease, including hedonic food intake and obesity. We performed a tripartite network analysis based on graph theory to test the hypothesis that microbiota-derived fecal metabolites are associated with connectivity of key regions of the brain's extended reward network and clinical measures related to obesity. METHODS: DTI and resting state fMRI imaging was obtained from 63 healthy subjects with and without elevated body mass index (BMI) (29 males and 34 females). Subjects submitted fecal samples, completed questionnaires to assess anxiety and food addiction, and BMI was recorded. RESULTS: The study results demonstrate associations between fecal microbiota-derived indole metabolites (indole, indoleacetic acid, and skatole) with measures of functional and anatomical connectivity of the amygdala, nucleus accumbens, and anterior insula, in addition to BMI, food addiction scores (YFAS) and anxiety symptom scores (HAD Anxiety). CONCLUSIONS: The findings support the hypothesis that gut microbiota-derived indole metabolites may influence hedonic food intake and obesity by acting on the extended reward network, specifically the amygdala-nucleus accumbens circuit and the amygdala-anterior insula circuit. These cross sectional, data-driven results provide valuable information for future mechanistic studies.