The nucleus accumbens (NAc) serves as a key neural substrate for aversive learning and consists of two distinct subpopulations of medium-sized spiny neurons (MSNs). The MSNs of the direct pathway (dMSNs) and the indirect pathway (iMSNs) predominantly express dopamine (DA) D1 and D2 receptors, respectively, and are positively and negatively modulated by DA transmitters via Gs- and Gi-coupled cAMP-dependent protein kinase A (PKA) signaling cascades, respectively. In this investigation, we addressed how intracellular PKA signaling is involved in aversive learning in a cell type-specific manner. When the transmission of either dMSNs or iMSNs was unilaterally blocked by pathway-specific expression of transmission-blocking tetanus toxin, infusion of PKA inhibitors into the intact side of the NAc core abolished passive avoidance learning toward an electric shock in the indirect pathway-blocked mice, but not in the direct pathway-blocked mice. We then examined temporal changes in PKA activity in dMSNs and iMSNs in behaving mice by monitoring Forster resonance energy transfer responses of the PKA biosensor with the aid of microendoscopy. PKA activity was increased in iMSNs and decreased in dMSNs in both aversive memory formation and retrieval. Importantly, the increased PKA activity in iMSNs disappeared when aversive memory was prevented by keeping mice in the conditioning apparatus. Furthermore, the increase in PKA activity in iMSNs by aversive stimuli reflected facilitation of aversive memory retention. These results indicate that PKA signaling in iMSNs plays a critical role in both aversive memory formation and retention.