We investigated the effect of light spectra on circadian rhythm by exogenous prolactin (PRL) by using light emitting diodes (LEDs): red, green, and purple. We injected PRL into live fish or treated cultured brain cells with PRL. We measured changes in the expressions of period 2 (Per2), cryptochrome 1 (Cry1), melatonin receptor 1 (MT1) mRNAs, and MT1 proteins, and in the plasma PRL, serotonin, and melatonin levels. After PRL injection and exposure to green LED light, MT1 expression and plasma melatonin levels were significantly lower, but the expressions of Per2 and Cry1 were significantly higher than others. Plasma serotonin after PRL injection and exposure to red LED light was significantly lower than others. These results indicate that injection of high concentration PRL inhibits melatonin, and inhibited melatonin regulates circadian rhythm via clock genes and serotonin. Thus, exogenous PRL regulates the circadian rhythm and light spectra influence the effect of PRL in goldfish.