Stress-induced reproductive dysfunction is frequently associated with increased glucocorticoid (GC) levels responsible for suppressed GnRH/LH secretion and impaired ovulation. Besides the major role of the hypothalamic kisspeptin system, other key regulators may be involved in such regulatory mechanisms. Herein, we identify dynorphin as a novel transcriptional target of GC. We demonstrate that only priming with high estrogen (E2) concentrations prevailing during the late prooestrus phase enables stress-like GC concentrations to specifically stimulate Pdyn (prodynorphin) expression both in vitro (GT1-7 mouse hypothalamic cell line) and ex vivo (ovariectomized E2-supplemented mouse brains). Our results indicate that stress-induced GC levels up-regulate dynorphin expression within a specific kisspeptin neuron-containing hypothalamic region (antero-ventral periventricular nucleus), thus lowering kisspeptin secretion and preventing preovulatory GnRH/LH surge at the end of the prooestrus phase. To further characterize the molecular mechanisms of E2 and GC crosstalk, chromatin immunoprecipitation experiments and luciferase reporter gene assays driven by the proximal promoter of Pdyn show that glucocorticoid receptors bind specific response elements located within the Pdyn promoter, exclusively in presence of E2. Altogether, our work provides novel understanding on how stress affects hypothalamic-pituitary-gonadal axis and underscores the role of dynorphin in mediating GC inhibitory actions on the preovulatory GnRH/LH surge to block ovulation.