Introduction: The nucleus paragigantocellularis lateralis (LPGi) is involved in the descending pain modulation. The neurostreoid, 17β-estradiol found in the PGi nucleus and modulates nociception by binding to estrogen receptors and also by allosteric interaction with NMDA receptors. In this study, the role of NMDA receptors in the 17β-estradiol-induced pain modulation was investigated by assessing the inflammatory pain responses changes after blockade of the LPGi nucleus’ NMDA receptors.  Methods: In order to study the antinociceptive effect of intra-LPGi microinjection of 17β-estradiol, a guide cannula was implanted into the right LPGi nucleus. 500 nl of drugs were administered 15 minutes prior to formalin (50 μl of 4%) injection. Then, formalin-induced paw jerking behaviour was recorded for 60 min. For assessing the role of the NMDA receptors in the pain modulation by 17β-estradiol, it was injected 15 min after the intra-LPGi administration of 0.5 nmol of AP5 (the NMDA receptor antagonist); and paw jerking frequency was recorded for 1 h. Results: The results of the present study showed that intra-LPGi injection of 0.8 μmol of 17β-estradiol attenuated the chronic phase (P<0.001) of paw jerking behaviour. AP5 significantly reduced the antinociceptive effect of intra-LPGi 17β-estradiol both in the acute (P<0.001) and in the chronic phase (P<0.001) of formalin test. Conclusion: According to the results of this study, it can be concluded that the analgesic effect of intra-LPGi injection of 17β-estradiol on the formalin-induced inflammatory pain might be mediated via NMDA receptors.