Despite the gut's longitudinal specialization for digestion and microbiome organization, most studies focus on the analysis of its end product, feces. To determine the metabolic and physiological functions of different sections of the gut, we aimed to define a comprehensive list of characteristic metabolites for the physiological gut sections and to quantify the selected pathways. We investigated the metabolic composition of seven different gut sections from four C57Bl/6N mice with nontargeted metabolite profiling using high-resolution NMR spectroscopy, which returned a comprehensive metabolite overview with a single analytical measurement per sample. Here we deliver a list of characteristic metabolites, describe metabolite changes along the gut, and highlight the site specificity for selected metabolite pathways. We find that the largest metabolic changes happen in the cecum, where the microbiome produces microbial metabolites. Furthermore, we show the evolution of bile acids along the gut and describe their site-specific conversion. We establish a metabolic basis for future investigations of metabolic perturbations, which can be introduced by dietary challenges or gene knockouts and provide valuable information for tailored study design and targeted sample collection.