Anemonefishes dominate the marine ornamental aquaculture production and coloration plays an important role in market price of the product. This study examined the effect of dietary esterified astaxanthin concentration and duration on skin coloration in spinecheek anemonefish, Premnas biaculeatus. The chromatophore physiology parameters examined were color, density, and diameter of carotenoid granules. Juveniles of 30 days post hatch (d.p.h.) were fed diets consisting of 23, 214, or 2350 ppm esterified astaxanthin for 115 d and the fish were sampled at 45, 120, and 145 d.p.h. Hue was significantly reduced with increasing time and astaxanthin concentration. Saturation fluctuated over time while luminosity was only affected by time. Carotenoid granule density was similar across the treatments: approximately 0.3 granules/µm2. Significant difference in carotenoid granule size was detected, with larger diameters at the higher astaxanthin treatment. Carotenoid granules showed significantly lower hues, higher saturation, and lower luminosity values for the higher astaxanthin concentration diets. This is indicative of higher concentration of carotenoids in the granules. Diets explicitly imparted different coloration in P. biaculeatus and the manifested color differences in the fish are result of increased carotenoid granule size and increased carotenoid concentration within granules. Astaxanthin concentration of 214 ppm imparts adequate coloration for high market value.