Background: Aspirin use is an effective strategy for the chemoprevention of colorectal cancer, even at low doses. However, in order to implement aspirin interventions, risk-benefit balances and biologic mechanisms need to be better defined; to further this aim, we used a metabolomics approach. Methods: We metabolically profiled 40 healthy, nonsmoking men and women ages 20 to 45 years enrolled in a randomized, double-blind, crossover trial of 325 mg aspirin/day over a period of 60 days. Gas and liquid chromatography-mass spectrometry were used to comprehensively profile participants' plasma samples after aspirin and placebo interventions. Results: A total of 363 metabolites, covering most human biochemical pathways, were measured. Compared with placebo-treated participants, plasma concentrations of the oncometabolite 2-hydroxyglutarate (R+S) decreased after aspirin treatment in both men and women (P = 0.005). This signal proved robust during 20-fold random splitting of the data using 80% of the samples in each split. We subsequently performed functional follow-up studies using targeted, enantiospecific detection in human colorectal cancer cell lines and observed an aspirin-induced reduction of (R)-2-hydroxyglutarate. We further showed that salicylate, the primary aspirin metabolite, inhibits the hydroxyacid-oxoacid transhydrogenase mediated production of (R)-2-hydroxyglutarate, thereby providing mechanistic evidence for the clinically observed effects of aspirin on total-2-hydroxyglutarate. Conclusions: Using a metabolomics approach with functional follow-up, we propose that a decrease in the oncometabolite (R)-2-hydroxyglutarate may identify an additional mechanism for aspirin or its metabolites in cancer prevention. Impact: Reduction of the oncometabolite (R)-2-hydroxyglutarate identifies a novel, non-COX-inhibition-mediated mechanism of aspirin.