MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEβ as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEβ mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEβ mutations. ► Treatment-emergent mutations in NAEβ confer resistance to MLN4924 ► Amino acid substitution A171T in the adenylate binding site is the most frequent mutation ► NAEβ mutants reduce inhibitor potency and decrease affinity of NEDD8-MLN4924 adduct ► A tight-binding pan-E1 inhibitor overcomes resistance in NAEβ mutant cells