Although it is widely accepted that steroid hormones, like estradiol (E2) can generate a sexually dimorphic response in neurons, little is known about the sexually differentiated signaling pathways that are influenced by them. Our previous studies have demonstrated that rapid non-genomic E2 actions on cAMP response element binding protein (CREB) are sexually differentiated in gonadotropin-releasing hormone (GnRH) neurons. However, the physiological importance of this sexual dimorphism still remains elusive. In this study, we have used GnRH neuron-specific CREB deleted mutant mice (GnRH-CREB KO) with or without global cAMP response element modulator (CREM) deletion to investigate the estrogen negative feedback on GnRH neurons in both sexes. In control mice of both sexes, gonadectomy increased plasma luteinizing hormone (LH) levels and these were then suppressed by acute E2 treatment. In female GnRH-CREB KO mice, basal levels of LH and the post-ovariectomy increment in LH were normal but the E2 induced rapid suppression of LH was significantly less effective. In contrast, in male GnRH-CREB KO mice, although basal LH concentrations and the response to E2 were normal, the post-gonadectomy increment in LH was significantly increased. These results demonstrate that CREB operates differently in GnRH neurons in females and males, and that CREB has important role in estrogen negative feedback in both sexes.