RF9, a reported antagonist of the mammalian gonadotropin-inhibitory hormone receptor, stimulates gonadotropin secretion in mammals. Recent studies have suggested that the stimulatory effect of RF9 on gonadotropin secretion relies on intact kisspeptin receptor (KISS1R) signaling, but the underlying mechanisms remain to be elucidated. Using Chinese Hamster Ovary cells stably transfected with KISS1R, we show that RF9 binds specifically to KISS1R, with a Kd of 1.6 × 10−5M, and stimulates an increase in intracellular calcium and inositol phosphate accumulation in a KISS1R-dependent manner, with EC50 values of 3.0 × 10−6M and 1.6 × 10−7M, respectively. RF9 also stimulated ERK phosphorylation, with a time course similar to that of kisspeptin-10. RFRP-3, the putative endogenous ligand for NPFFR1, did not stimulate inositol phosphate accumulation or pERK, nor did it alter responses to of kisspeptin-10 or RF9. In agreement with these in vitro data, we found that RF9 stimulated a robust LH increase in Npffr1−/− mice, similar to that in wild-type littermates, whereas the stimulatory effect of RF9 was markedly reduced in Kiss1r−/− and double Kiss1r−/−/Npfrr1−/− mice. The stimulatory effect of RF9 on LH secretion was restored by the selective rescue of Kiss1r expression in GnRH neurons, in Kiss1r−/−T mice. Taken together, our study demonstrates that RF9 acts primarily as a KISS1R agonist, but not as an allosteric modulator, to stimulate LH secretion. Our findings raise questions regarding the utility of RF9 for assessing NPFF1R function and de-emphasize a predominant role of this signaling system in central regulation of reproduction.