The gamma-carotenoids, such as myxol and saproxanthin, have a high potential to be utilized in nutraceutical and pharmaceutical industries for their neuro-protective and antioxidant effects. CrtD is involved in the production of gamma-carotenoids by desaturating the C3'-C4' position of 1'-OH-gamma-carotenoid. We determined the crystal structure of CrtD from Nonlabens dokdonensis DSW-6 (NdCrtD), the first structure of CrtD family enzymes. The NdCrtD structure was composed of two distinct domains, an FAD-binding domain and a substrate-binding domain, and the substrate-binding domain can be divided into two subdomains, a Rossmann fold-like subdomain and a lid subdomain. Although the FAD-binding domain showed a structure similar to canonical FAD-containing enzymes, the substrate-binding domain exhibited a novel structure to constitute a long and hydrophobic tunnel with a length of similar to 40 angstrom. The molecular docking-simulation reveals that the tunnel provides an appropriate substrate-binding site for the carotenoid such as 1'-OH-gamma-carotene with a length of similar to 35 angstrom. We could predict residues related to recognize the 1'-hydroxyl group and to stabilize the hydrophobic end without hydroxyl group. Moreover, we suggest that the flexible entrance loop may undergo an open-closed formational change during the binding of the substrate. (C) 2015 Elsevier Inc. All rights reserved.