As cancer cells undergo metabolic reprogramming in the course of tumorigenesis, targeting energy metabolism represents a promising strategy in cancer therapy. Among various metabolic enzymes examined, pyruvate kinase M2 type (PKM2) has received much attention in light of its multifaceted function in promoting tumor growth and progression. In this study, we reported the development of a novel irreversible inhibitor of PKM2, compound 1, that exhibits a differential tumor-suppressive effect among an array of cancer cell lines. We further used a clickable activity-based protein profiling (ABPP) probe and SILAC coupled with LC-MS/MS to identify the Cys-317 and Cys-326 residues of PKM2 as the covalent binding sites. Equally important, compound 1 at 10 mg/kg was effective in suppressing xenograft tumor growth in nude mice without causing acute toxicity by targeting both metabolic and oncogenic functions. Together, these data suggest its translational potential to foster new strategies for cancer therapy.