Mitochondrial beta-oxidation involves a family of flavoproteins that introduce a C-C double bond into their fatty acyl-CoA substrates. Deficiencies of these acyl-CoA dehydrogenases lead to fatty acid oxidation disorders involving life-threatening episodes of metabolic derangement. This review focuses on the medium chain acyl-CoA dehydrogenase as the best-understood member of its class. The crystal structure of the enzyme and salient features of its substrate specificity and mechanism of action are summarized. The surprising observation of a catalytically essential amino acid residue that nevertheless is not conserved in the acyl-CoA dehydrogenase family is discussed.