Ubiquitin-conjugating Enzyme E2I (UBE2I), also known as ubiquitin-conjugating enzyme 9 (Ubc9), is a ubiquitously expressed protein with a predicted molecular weight of 20 kDa. Human UBE2I/Ubc9 shares 100% amino acid sequence identity with the mouse and rat orthologs. UBE2I/Ubc9 catalyzes the addition of the ubiquitin-like protein SUMO to target proteins. SUMO is transferred from a ubiquitin-like activating (E1) enzyme heterodimer consisting of SAE1 and UBA2 to Cys93 of UBE2I/Ubc9. In contrast to most ubiquitin-conjugating (E2) enzymes that function in a complex with ubiquitin ligases (E3s), UBE2I/Ubc9 can interact directly with protein substrates and may also play a role in substrate recognition. UBE2I/Ubc9 mediates the SUMOylation of a variety of proteins including RanGAP1, HDAC4 and PML. Post-translational modifications of UBE2I/Ubc9, such as auto-SUMOylation at Lys14 or Ser71 phosphorylation by CDC2/CDK1, alter UBE2I/Ubc9 catalytic activity and target protein recognition. UBE2I/Ubc9-dependent SUMOylation reduces the levels of the stem cell marker Nanog, implicating it in embryonic stem cell pluripotency maintenance. SUMOylation also regulates the protein levels of tumor suppressors and oncogenes, and UBE2I/Ubc9 dysregulation is thought to contribute to the pathogenesis of human cancers.