Ovulation is triggered by the preovulatory rise of gonadotropins, which is in turn elicited by the preceding increase in circulating estrogen. Kisspeptins, ligands of GPR54 encoded by KiSS-1 gene, have emerged as potent stimulators of GnRH/LH secretion, and KiSS-1 neurons at the anteroventral periventricular (AVPV) nucleus have been shown to be involved in the generation of preovulatory LH surge, estrogen being a potent elicitor of KiSS-1 gene expression selectively at AVPV. Whether, in addition to transcriptional effects, estrogen influences other aspects of kisspeptin-induced GnRH/LH release in the female remains unexplored. We provide herein evidence for the specific roles of estrogen receptor (ER) and ER in the modulation of LH responses to kisspeptin and the generation of the preovulatory surge. Selective blockade of ER in cyclic females blunted LH responses to kisspeptin, eliminated the endogenous preovulatory rise of LH, and blocked ovulation. In contrast, antagonization of ER failed to cause major changes in terms of LH surge and ovulatory rate, but significantly augmented acute LH responses to kisspeptin. Notably, defective LH secretion and ovulation following ER blockade were not observed after GnRH stimulation, which elicited maximal acute (<2-h) LH responses regardless of ER /ER signaling. In addition, net LH secretion in response to kisspeptin was decreased by ovariectomy and increased following selective activation of ER, but not ER. Altogether, our data document the prominent positive role of ER in the regulation of GnRH/LH responsiveness to kisspeptin and, thereby, ovulation. In addition, our results disclose the putative function of ER as negative modifier of GnRH/LH response to kisspeptin, a phenomenon that might contribute to partially restraining LH secretion at certain physiological states.