In order to investigate how photoperiod affects the expression of KiSS-1/G-protein-coupled receptor 54 (GPR54), the hypothalamus, the ovaries and testicles of the striped hamster were studied following photoperiod treatment and melatonin injection. We found that the melatonin concentration in the blood of the striped hamster was consistent with the treated photoperiods and that the photoperiod had significant effects on KiSS-1 expression, but the effect of photoperiod on GPR54 expression differed among tissues and sexes. The relative expression of KiSS-1 in the gonads of the male and female striped hamster was significantly higher than that in the hypothalamus, while the difference in GPR54 between the gonads and hypothalamus was inconsistent between the male and female striped hamster. In the striped hamster injected with melatonin, the expression level of KiSS-1 in the gonads and hypothalamus of the male and female striped hamster was significantly decreased; the expression level of GnRH in the gonads was also significantly decreased; the expression of GPR54 in the hypothalamus was increased. In the hypothalamus, a significant positive correlation existed between KiSS-1 and GnRH expression, while a negative correlation was observed between GPR54 and GnRH expression. These results suggest that the photoperiod-melatonin-KiSS-1-GnRH pathway plays an important role in the seasonal reproduction mechanism of the striped hamster. Additionally, photoperiod or melatonin may not be a direct regulatory factor of GPR54 expression.