Previous studies have demonstrated that morphine, administered systemically or directly into the PAG, produces a significantly greater degree of antinociception in males in comparison to females. As the midbrain periaqueductal gray (PAG) and its descending projections to the rostral ventromedial medulla (RVM) constitute an essential neural circuit for opioid-based analgesia, the present studies were conducted to determine if sex differences in the anatomical organization of the PAG-RVM pathway, and its activation during persistent inflammatory pain, could account for sex-based differences in opioid analgesia. In the rat, retrograde tracing was combined with Fos immunocytochemistry to investigate sexual dimorphism in the organization of the PAGRVM circuit and its activation by persistent inflammatory pain induced by intraplantar injection of complete Freund’s adjuvant (CFA). The ability of morphine to suppress the activation of the PAG-RVM circuit was also examined. Sexually dimorphic retrograde labeling was observed within the dorsomedial and lateral/ventrolateral PAG at all rostrocaudal levels, with females having significantly more PAG-RVM output neurons in comparison to males. While no sex differences were noted in the activation of the PAGRVM circuit by persistent inflammatory pain, significantly more double labeled cells were found in males in comparison to females. Systemic administration of morphine significantly suppressed CFA-induced Fos in the PAG in males only. The results of these studies demonstrate that both the anatomical organization, and functional activation, of the PAG-RVM circuit is sexually dimorphic, and may provide the anatomical substrate for sex-based differences in morphine analgesia.