Acyl-CoA oxidases (ACOXs) play important roles in lipid metabolism, including peroxisomal fatty acid β-oxidation by the conversion of acyl-CoAs to 2-trans-enoyl-CoAs. The yeast Yarrowia lipolytica can utilize fatty acids as a carbon source and thus has extensive biotechnological applications. The crystal structure of ACOX3 from Y. lipolytica (YlACOX3) was determined at a resolution of 2.5 A. It contained two molecules per asymmetric unit, and the monomeric structure was folded into four domains; Nα, Nβ, Cα1, and Cα2 domains. The cofactor flavin adenine dinucleotide was bound in the dimer interface. The substrate-binding pocket was located near the cofactor, and formed at the interface between the Nα, Nβ, and Cα1 domains. Comparisons with other ACOX structures provided structural insights into how YlACOX has a substrate preference for short-chain acyl-CoA. In addition, the structure of YlACOX3 was compared with those of medium- and long-chain ACOXs, and the structural basis for their differences in substrate specificity was discussed.