Ubiquitin-conjugating enzymes (E2s) govern key aspects of ubiquitin signaling. Emerging evidence suggests that the activities of E2s are modulated by posttranslational modifications; the structural underpinnings, however, are largely unclear. Here, we unravel the structural basis and mechanistic consequences of a conserved autoubiquitination event near the catalytic center of E2s, using the human anaphase-promoting complex/cyclosome-associated UBE2S as a model system. Crystal structures we determined of the catalytic ubiquitin carrier protein domain combined with MD simulations reveal that the active-site region is malleable, which permits an adjacent ubiquitin acceptor site, Lys⁺⁵, to be ubiquitinated intramolecularly. We demonstrate by NMR that the Lys⁺⁵-linked ubiquitin inhibits UBE2S by obstructing its reloading with ubiquitin. By immunoprecipitation, quantitative mass spectrometry, and siRNA-and-rescue experiments we show that Lys⁺⁵ ubiquitination of UBE2S decreases during mitotic exit but does not influence proteasomal turnover of this E2. These findings suggest that UBE2S activity underlies inherent regulation during the cell cycle. Ubiquitin-conjugating enzymes (E2s) act at the heart of a catalytic cascade that modifies proteins with ubiquitin and regulates countless physiological processes. Liess et al. elucidate the structural basis of a regulation mechanism that seems to be conserved in 25% of human E2s, using UBE2S as a model system.