In contrast to soluble acyl-ACP desaturases from plants, little is known about the structure-guiding principle underlying substrate specificity and regioselectivity of membrane-bound acyl-CoA desaturases from animals, mainly due to lack of the three-dimensional structure information. Here we report identification of two homologous membrane-bound acyl-CoA Δ9 desaturases (ChDes9-1 and ChDes9-2) from the marine copepod Calanus hyperboreus that accumulates more than 90% of total storage lipids in the form of wax esters. ChDes9-2 is a common Δ9 desaturase with substrate specificity to long chain fatty acid 18:0, while ChDes9-1 is a new type of Δ9 desaturase introducing a Δ9 double bond into a wide range of very long chain fatty acids ranging from 20:0 to 26:0. Reciprocal domain swapping and site-directed mutagenesis guided by the membrane topology revealed that presence or absence of an amphipathic and bulky residue, tyrosine, in the middle of the second transmembrane domain was important in determining the substrate specificity of the two desaturases. To examine the mechanistic structure for the substrate specificity, tyrosine-scanning mutagenesis was employed to systematically substitute the residues in the transmembrane domain of the very long chain desaturase. The results showed that the transmembrane domain formed an α-helix structure probably involved in formation of the substrate-binding pocket and the corresponding residue of the tyrosine likely resided at the critical position within the pocket mediating the interaction with the substrates, thereby specifying the chain length of the substrates.