Male peacock (Pavo cristatus) tail feathers have an eyespot pattern with an inconspicuous black or dark blue center surrounded by brilliant, structural colors, such as blue, light brown, and yellow-green. Under ultraviolet A (UVA), the central part of the eyespot reflects UVA better than the surrounding parts. Herein, I examined various areas of eyespots on paraffin sections of feathers using an optical microscope, and characterized positional relationships between barbs and barbules. These analyses confirmed that barbules in the central part of the eyespot are in a horizontal position with respect to the barb, and that light transmission from the central part is less than that from the other parts. In addition, I compared microstructures of barbules in the central part of eyespot with those in surrounding areas using transmission electron microscope analysis. The melanin rods in the barbules reflecting yellow-green color comprise several ordered lattice structures. In contrast, melanin rods in the central part of the eyespot were only distributed in 1–3 layers on a part of the front side of the barbules. I also demonstrated that keratin structures of barbules are homogeneous in the central part of the eyespot, but have fibrous structures with many voids in the yellow-green parts. Collectively, the present observations suggest that feathers in the central part of the eyespot reflect UVA depending on the direction of irradiation, and these properties are governed by configurations of barbules relative to barbs, melanin rod distributions, and the presence of keratin structures with gaps.