Introduction: The 17β-estradiol acts as a neurosteroid in the brain and modulates nociception by binding to the estrogen receptors and also by allosteric interaction with other membrane-bound receptors like glutamate receptors. Paragigantocellularis lateralis nucleus (LPGi) is one of the important brain regions implicated in the pain modulation. So, this study was designed to evaluate the possible involvement of the membrane-bound AMPA receptors of LPGi nucleus in the pain modulatory effect of intra-LPGi 17β-estradiol in the male rats. Methods: In order to study the pain modulatory effect of intra-LPGi microinjection of 17β-estradiol, cannulation into the LPGi nucleus was performed. Then, 500 nl of saline, 17β-estradiol and CNQX- the AMPA receptor antagonist- were unilaterally administered into the right LPGi by injection cannula and Hamilton syringe. In addition, for assessing the role of the AMPA receptors in the pain modulation by 17β-estradiol, 17β-estradiol was injected 15 min after the intra-LPGi administration of CNQX. Then, 50 μl of 4% formalin was subcutaneously injected into the plantar surface of contralateral hind paw and the number of paw jerking behavior was observed for 60 min. Results: The results showed that intra-LPGi injection of 0.8 μmol of 17β-estradiol attenuated the chronic phase (P<0.001) of paw jerking behaviour. CNQX significantly prevented the antinociceptive effect of intra-LPGi 17β-estradiol both in the acute (P<0.05) and in the chronic phase (P<0.001) of formalin test. Conclusion: Considering the results of this study, it can be concluded that the analgesic effect of intra-LPGi injection of 17β-estradiol might be mediated via AMPA receptors.