Photoperiodic signals are transduced into the rhythmic release of the pineal hormone melatonin, which synchronizes reproduction with season. Novel peptides such as the RF-related peptide (RFRP) are considered to play a role in the seasonal regulation of the reproductive axis. The expression of RFRP-neurons in the mediobasal hypothalamus is strongly down-regulated by melatonin in short day (SD) conditions. RFRP-3 has been shown to regulate reproductive activity but whether RFRP-3 is inhibitory or excitatory depends on gender and species. It is not yet clear whether RFRP-3 exerts its actions directly on GnRH-neurons or upstream i.e. on kisspeptin-neurons. In this study we aimed to investigate photoperiod-dependent variations in the RFRP system in male and female Syrian hamster, by determining the number of RFRP-immunoreactive neurons, the density of RFRP-fiber projections, as well as the expression of the RFRP receptor GPR147. We found that the neuroanatomical distribution of RFRP-immunoreactive fibers and GPR147 mRNA are similar in male and female Syrian hamster. However, both RFRP-neuronal expression and GPR147 mRNA levels are significantly higher in female than in male. GPR147 is mainly expressed in the preoptic area, anteroventral-periventricular nucleus (AVPV), suprachiasmatic nucleus, paraventricular nucleus, ventromedial hypothalamus, habenular nucleus and the arcuate nucleus (ARC). GPR147 mRNA levels as well as RFRP-neuronal expression is down-regulated in SD, however with a stronger decrease in female than in male. The density of RFRP-fiber projections was studied in areas expressing GnRH- (preoptic area) and kisspeptin-neurons (AVPV and ARC). Interestingly, RFRP-fiber density in the AVPV was found to be higher in females in SD. These findings show that RFRP and its receptor GPR147 are regulated by photoperiod in both male and female Syrian hamster. However, our results suggest that the RFRP system is more sensitive in female with a distinct role in the AVPV, probably in regulation of the pre-ovulatory LH surge.