During the breeding season, dynamic changes in body coloration are regularly observed in the male brown tree frog Buergeria robusta. This study investigated the hypothesis that this sexual dichromatism in male B. robusta is mediated through hormonal regulation. Frogs were exogenously injected with testosterone (T) or estradiol (E2). This manipulation revealed that the body coloration (hue, brightness, and saturation) of the male frog increased significantly (i.e., the brilliant yellow color developed) in response to T but not in response to E2. Concurrently, the levels of expression of brain-derived neurotrophic factor (BDNF) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the pituitary gland were reduced in frogs whose coloration was pale brown on a yellow background. In particular, the weakest expressions of BDNF, PACAP, and PACAP type II receptors (VPAC-1R) were found in male frogs with a brilliant yellow body color during the breeding season regardless of background color. These changes may decrease α-melanocyte-stimulating hormone production associated with the PACAP receptors (VPAC-1R), resulting in the aggregation of black pigment in melanophores and the production of a brilliant yellow body color. The effects of hormones on skin coloration were further examined in isolated skin in vitro. The results of this investigation showed that the dispersion of xanthophores was stimulated by T or prolactin (PRL) and that the melanophores were aggregated by melatonin (MEL) but not by E2. Furthermore, yellow pigments in the xanthophores were significantly dispersed following the PRL+T treatment. In the T+MEL, PRL+MEL, and T+PRL+MEL treatments, xanthophores were dispersed, and melanophores were aggregated and subsequently moved to the low spongiosum layer of the dorsal skin, causing the increase in yellow coloration. These results reveal that multiple hormones play major roles in the regulation of the brilliant yellow coloration of male B. robusta by high plasma T during the breeding season.