The involvement of estrogen in pain has been investigated in many ways. However the specific role played by estrogen receptors remains elusive. Estrogen receptors alpha and beta mediate different physiological functions. For example, estrogen receptor beta is more closely related to non-reproductive effects than the alpha subtype is. To verify the involvement of estrogen receptor beta on acute and persistent pain as well as on endogenous pain inhibitory mechanisms, hotplate and formalin tests were carried out in wild type (WT) and estrogen receptor beta knockout (ERbeta KO) mice of both sexes. Ovariectomies followed by estrogen and progesterone replacement were performed in female groups to insure comparable sex hormone levels. We found that nociceptive responses are lower in ERbeta KO female than in WT female mice during the interphase and early tonic phase II of the formalin test but not during acute and late tonic phases. Moreover, behavioral and spinal (c-Fos) differences were only observed in females. ERbeta KO females had lower c-Fos expression in laminae I-II and IV-V of the spinal cord than WT females. These results suggest that estrogen, through its actions on ERbeta, dampens the efficacy of endogenous pain modulation mechanisms during the interphase and/or inflammation process in the early phase II, triggering an increase in spinal nociceptive neuronal activity. This confirms our previous observations that estrogen specifically influences nociceptive responses during the interphase of the formalin test and demonstrates a role for ERbeta on endogenous pain modulation systems.