The rostral anterior cingulate cortex (rACC) is a key structure of pain affect. Whether and how estrogen in the rACC regulates pain-related negative emotion remains unclear. Behaviorally, using formalin-induced conditioned place aversion (F-CPA) in rats, which is believed to reflect the pain-related negative emotion, we found that estrogen receptor (ER) inhibitor ICI 182, 780 (ICI, 7α,17β-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol) or inhibitor of aromatase androstatrienedione into the rACC completely blocked F-CPA in either sex. An analogous effect was also observed in ovariectomy rats. Furthermore, exogenous estrogen in the absence of a formalin noxious stimulus was sufficient to elicit CPA (E-CPA) in both sexes by activating the membrane estrogen receptors (mERs) and N-methyl-D-aspartic acid (NMDA) receptors (NMDARs). Electrophysiologically, we demonstrated that estrogen acutely enhanced the glutamatergic excitatory postsynaptic currents (EPSCs) in rACC slices by increasing the ratio of NMDA–EPSCs to α-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid –EPSCs and presynaptic glutamate release. Interestingly, a brief exposure to estrogen elicited a persistent enhancement of NMDA–EPSCs, and this NMDA–long-term potentiation required the activation of the mERs, protein kinase A and NMDAR subunit NR2B. Finally, estrogen induced rapid dendritic spine formation in cultured rACC neurons. These results suggest that estrogen in the rACC, as a neuromodulator, drives affective pain via facilitating NMDA receptor-mediated synaptic transmission.