The mechanisms through which estradiol (E2) regulates gonadotropin-releasing hormone (GnRH) neurons to control fertility are unclear. Previous studies have demonstrated that E2 rapidly phosphorylates cAMP response element-binding protein (CREB) in GnRH neurons in vivo. In the present study, we used GnRH neuron-specific CREB-deleted mutant mice [GnRH-CREB knock-outs (KOs)] with and without global cAMP response element modulator (CREM) deletion (global-CREM KOs) to investigate the role of CREB in estrogen negative feedback on GnRH neurons. Evaluation of GnRH-CREB KO mice with and without global CREM deletion revealed normal puberty onset. Although estrus cycle length in adults was the same in controls and knock-out mice, cycles in mutant mice consisted of significantly longer periods of diestrus and less estrus. In GnRH-CREB KO mice, basal levels of luteinizing hormone (LH) and the postovariectomy increment in LH were normal, but the ability of E2 to rapidly suppress LH was significantly blunted. In contrast, basal and postovariectomy LH levels were abnormal in GnRH-CREB KO/global-CREM KO mice. Fecundity studies showed that GnRH-CREB KO with and without global CREM deletion were normal up to ∼9 months of age, at which time they became prematurely reproductively senescent. Morphological analysis of GnRH neurons revealed a significant reduction (p < 0.01) in GnRH somatic spine density of GnRH-CREB KO mice compared to control females. These observations implicate CREB within the GnRH neuron as an important target for E2's negative feedback actions. They also indicate that the rapid modulation of CREB by E2 is of physiological significance in the CNS.