Background Studies have shown that N-methyl-D-aspartate (NMDA) receptors play a critical role in pain processing at different levels of the central nervous system. Methods In this study, we used adult Wistar rats to examine gender differences in the effects of NR2B NMDA antagonism at the level of the anterior cingulate cortex in phasic pain, and in the first and second phases of a formalin test. Rats underwent stereotactic surgery for cannula implantation in the anterior cingulate cortex. After recovery, paw withdrawal latency to a noxious thermal stimulus was assessed. Rats were also subjected to a formalin pain test whereby 60 μL of 5% formalin was injected into the right hind paw. Results Female and male rats that received Ro 25-6981, an NR2B antagonist, before formalin injection showed significantly reduced pain responses to the formalin test compared with saline-injected control rats (P < 0.05). No gender differences in phasic pain responses were found in rats treated with Ro 25-6981. Conclusion These results suggest that cortical antagonism of the NR2B subunit reduces inflammatory pain levels in both genders of rat.