The intestines are key for the absorption of nutrients and water as well as drug metabolism, and it is well known that there are clear differences in the expression profile of drug metabolism enzymes along the intestinal tract. Yet, only a few studies have thoroughly investigated regional differences in human intestinal drug metabolism. In this study, we evaluated phase I and phase II metabolism in matched human ileum and colon precision-cut intestinal slices (PCIS). To this end, human PCIS were incubated for 3 h with testosterone (TT) and 7-hydroxycoumarin (7-HC) to examine phase I and phase II metabolism, respectively. Metabolite formation was assessed by high-performance liquid chromatography (HPLC) analysis. Our results demonstrated that androstenedione, 6β-hydroxytestosterone, 2β-hydroxytestosterone, and 7-HC sulfate, were predominantly formed in the ileum, while 15α-hydroxytestosterone and 7-HC glucuronide were mainly produced in the colon. Moreover, we also observed sex differences in phase II metabolite formation, which appeared to be higher in males as compared to females. Taken together, we demonstrated that phase I metabolism predominantly occurs in ileum PCIS, while phase II metabolism mostly takes place in colon PCIS. Moreover, we revealed that human PCIS can be used to study both regional and sex differences in intestinal metabolism.