USP20, one of deubiquitinating enzymes (DUBs) belonging to the subfamily of ubiquitin-specific protease (USP), regulates ubiquitin-mediated protein degradation. So far, USP20 has been identified as a binding protein and a regulator of hypoxia-inducible factor (HIF)-1α, β-adrenergic receptor, and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). In order to investigate other biological functions of USP20 with its novel substrates, we searched for putative substrates through two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis. We found several putative substrates, some of which are related to cancer metabolism or neural disorders. Among these, the pyruvate kinase isoenzyme M2 (PKM2) had a high identity score. Most cancer cells contain a specific metabolic pathway, referred to as the Warburg effect. One well-known function of PKM2 is a main regulator in cancer metabolic pathways, and PKM2 promotes the Warburg effect and tumor growth. In addition, both PKM2 and HIF-1α upregulate the expression of target genes. From this evidence, it is expected that USP20 would be associated with the metabolic pathway through the regulation of PKM2 ubiquitination. Despite various roles of DUBs, the biological functions of USP20 in cellular mechanisms are poorly understood. Herein, we investigated the inter-action between PKM2 and USP20. Our results suggest a new molecular pathway in cancer metabolism through the regulation of PKM2.