We have previously shown that DNIC is stronger in male rats compared to female rats and that the efficient DNIC in males is associated with a stronger functional connectivity between rACC and PAG. In this study, we investigated the nature of interactions between rACC and PAG in the context of our DNIC paradigm. First, we examined whether rACC is a critical brain region for mediating DNIC by inactivating rACC with lidocaine. The temporary inactivation of rACC in males led to a DNIC response similar to that observed in females. We then investigated whether the DNIC phenomena we observed involved endogenous opioid mechanisms. Systemic treatment with naloxone significantly attenuated the DNIC response in males and eliminated sex differences. To investigate whether PAG plays a role in mediating endogenous opioid effects, we microinjected naloxone directly into PAG of males. Consistent with our prediction, this treatment significantly attenuated the DNIC response in males. Moreover, microinjection of morphine directly into the PAG of females significantly enhanced the DNIC response to an extent similar to that observed in males. We then conducted a preliminary chemogenetic experiment in which we selectively inhibited rACC neurons projecting to PAG in male rats. DNIC responses in males transfected with DREADDi were significantly blunted. Collectively, these results support our hypothesis that strong rACC to PAG connectivity, which involves opioid processing in PAG, is required for efficient DNIC. We also showed that exercise improves DNIC in both male and female rats, suggesting exercise as a potential tool to increase DNIC responses. NIH-NIA RO1 AG073126 (JYR) and NIH-NIDCR R21 DE027808 (JYR).