Physiological color change in camouflage and mating is widespread among fishes, but little is known about the regulation of such temporal changes in nuptial coloration and particularly concerning female coloration. To better understand regulation of nuptial coloration we investigated physiological color change in female two-spotted gobies (Gobiusculus flavescens). Females of this species develop an orange belly that acts as an ornament. The orange color is caused by the color of the gonads combined with the chromathophore based pigmentation and transparency of the skin. Often during courtship and female-female competition, a rapid increase in orange coloration, in combination with lighter sides and back that increases skin and body transparency, gives the belly an intense 'glowing' appearance. To understand how this increased orange coloration can be regulated we analysed chromatic and transparency effects of neurohumoral agents on abdominal skin biopsies in vitro. We found prolactin and alpha-melanocyte stimulating hormone (MSH) to increase orange coloration of the skin. By contrast, melatonin and noradrenaline increased skin transparency, but had a negative effect on orange coloration. However, mixtures of melatonin and MSH, or melatonin and prolactin, increased both orange coloration and transparency. This effect mimics the chromatic 'glow' effect that commonly takes place during courtship and intra sexual aggression. Notably, not only epidermal chromatophores but also internal chromatophores lining the peritoneum responded to hormone treatments. There were no chromatic effects of the sex steroids 17beta-estradiol, testosterone or 11-ketotestosterone. We hypothesize that similar modulation of nuptial coloration by multiple hormones may be widespread in nature.