Biased mating due to female preferences towards certain traits in males is a major mechanism driving sexual selection, and may constitute an important evolutionary force in organisms with sexual reproduction. In birds, although the role of male ornamentation, plumage coloration, genetic dissimilarity, and body size have on mate selection by females have been examined extensively, few studies have clarified exactly how these characteristics affect female mate preferences. Here, we show that testosterone (T)-dependent male attractiveness enhances female preference for males of a polygamous species, the Japanese quail. A significant positive correlation between female mating preference and circulating T in the male was observed. The cheek feathers of attractive males contained higher levels of melanin and were more brightly colored. The ability of females to distinguish attractive males from other males was negated when the light source was covered with a sharp cut filter (cutoff; < 640 nm). When females were maintained under short-day conditions, the expression of retinal red-sensitive opsin decreased dramatically and they became insensitive to male attractiveness. Our results showed that female preference in quail is strongly stimulated by male feather coloration in a T-dependent manner and that female birds develop a keen sense for this coloration due to upregulation of retinal red-sensitive opsin under breeding conditions.