E3 ligases drive the specificity of ubiquitin (UB) and UB-like (UBL) protein ligation. Diverse E3 structures provide distinct mechanisms achieving timely and accurate formation of covalent bonds between targeted proteins and C-terminus of UB or a UBL. Meanwhile, cellular regulation also depends on E3 ligase inhibition, activation, target prioritization and selection between UB and UBLs. This chapter summarizes recent studies revealing remarkable mechanisms of action and regulation of major classes of eukaryotic UB ligases – cullin-RING, HECT and RBR E3s – with additional focus on distinct activities of small-molecule probes that have been developed to perturb these systems for pharmaceutical and biochemical discovery purposes.