Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function analysis, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, and computational modeling, to identify a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted a-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, we find that enzyme mislocalization underlies the metabolic deficiency syndrome of patients bearing specific mutations that disrupt the structure of an a-helical membrane binding region of VLCAD.