Estradiol is a female hormone required for maintaining pregnancy and developing follicles in the ovary. Estradiol has been shown to perform a variety of physiological activities, including pain reduction. In this study, we tested the hypothesis that estradiol exerts antinociceptive effects in a rat model of inflammatory hyperalgesia. We established a subacute hyperalgesia model using plantar injection of Freund's complete adjuvant (FCA) in Sprague-Dawley rats. We administered estradiol every 24 h, beginning 12 h after FCA administration, and used the plantar test to determine its effect on hyperalgesia. To determine the mechanism of action of estradiol, we evaluated the role of the opioid antinociceptive system using naloxone and the role of the descending pain inhibitory system using the α-2-receptor antagonist yohimbine and the serotonin receptor antagonist methysergide. Administration of FCA induced hyperalgesia, which was significantly reduced by estradiol treatment compared to controls. Moreover, this effect was not antagonized by naloxone, but was attenuated by α-2-receptor and serotonin-receptor antagonists. Estradiol is known to perform a variety of physiological functions. Our findings suggest that one such function is antinociception via an interaction with α-2 receptors and serotonin receptors.