Acarbose, an alpha-glucosidase inhibitor used in treating type 2 diabetes, impairs complex carbohydrate digestion and absorption and extends life span in mice (without a requisite reduction in food intake). To assess sex-differential effects coincident with calorie restriction versus a nonrestricted longevity enhancing intervention, we evaluated the metabolite profiles (by liquid chromatography–mass spectroscopy) from livers and cecal contents of C57BL/6J mice (n = 4/sex/group), which were maintained for 10 months under one of the three diet treatments: ad libitum control diet (CON), ad libitum control diet containing 0.1% acarbose (ACA), or 40% calorie restriction using the control diet (CR). Principal component analysis revealed sex-differential profiles with ACA in livers. Of the identified metabolites (n = 621) in liver, CR significantly altered ~44% (males:187↑/131↓, females:74↑/148↓) compared with CON, in contrast with ACA (M:165↑/61↓, F:52↑/60↓). Dissimilarity in ACA-F liver metabolites was observed for ~50% of common metabolites from ACA-M and CR-M/F. CR resulted in fewer significant cecal metabolite differences (n = 615 metabolites; M:86↑/66↓, F:51↑/48↓ vs CON), relative to ACA treatment (M:32↑/189↓, F:36↑/137↓). Metabolomic profiling identifies sex-differential and tissue-specific effects with amino acid metabolism sub-pathways including those involving tryptophan, branch-chain and sulfur amino acids, and the urea cycle, as well as bile acid, porphyrin, and cofactor metabolism pathways.