Abstract Macroautophagy is one of two major degradation systems in eukaryotic cells. Regulation and control of autophagy is often achieved through the presence of short peptide sequences called LC3 interacting regions (LIR) in autophagy-involved proteins. Using a combination of new protein-derived activity-based probes, protein modelling and X-ray crystallography, we identified a non-canonical LIR motif in the human E2 enzyme responsible for LC3 lipidation, ATG3. The LIR motif is present in the flexible region of ATG3 and adopts an uncommon β-sheet structure binding to the backside of LC3. We show that the β-sheet conformation is crucial for its interaction with LC3. In cellulo studies provide evidence that LIR ATG3 is required for LC3 lipidation and ATG3∼LC3 thioester formation. Removal of LIR ATG3 negatively impacts the rate of thioester transfer from ATG7 to ATG3. Abstract Figure