Menopausal and postmenopausal women, characterized by a significant reduction in ovarian hormones, have a high prevalence of chronic pain with great pain intensity. However, the underlying mechanism of hyperalgesia induced by ovarian hormone withdrawal remains poorly understood. Here, we report that decreases in the activity and excitability of GABAergic neurons in the dorsal raphe nucleus (DRN) are associated with hyperalgesia induced by ovariectomy in mice. Supplementation with 17β-estradiol, but not progesterone, is sufficient to increase the mechanical pain threshold in ovariectomized (OVX) mice and the excitability of DRN GABAergic (DRN GABA ) neurons. Moreover, activation of the DRN GABA neurons projecting to the lateral parabrachial nucleus was critical for alleviating hyperalgesia in OVX mice. These findings show the essential role of DRN GABA neurons and their modulation by estrogen in regulating hyperalgesia induced by ovarian hormone withdrawal, providing therapeutic basis for the treatment of chronic pain in physiological or surgical menopausal women.