Pituitary adenylate cyclase-activating polypeptide (PACAP) and its preferred receptor PAC1 have been associated with stress responses and psychiatric disorders such as anxiety, depression, and post-traumatic stress disorder as shown by human genetic and animal model studies. We have recently shown that a single intraperitoneal administration of a small-molecule PAC1 antagonist PA-915 improved depressive-like behaviors in repeated social defeat stress (RSDS) mice. However, it remains unknown where and how PACAP-PAC1 signaling is involved in the regulation of depressive-like behaviors in the brain. In this study, we focused on the medial prefrontal cortex (mPFC) which is known to play a pivotal role in emotional processing and stress response, and performed electrophysiological, behavioral, and pharmacological analyses. We found that the decrease in the excitation and inhibition (E/I) ratio of the layer V pyramidal neurons in RSDS mice was significantly restored by the intraperitoneal treatment of PA-915. We also found that local administration of PA-915 in the mPFC improved depression-like behaviors in RSDS mice. These results indicate that PACAP-PAC1 signaling in the mPFC may modulate depressive-like behaviors by rebalancing the activities of pyramidal neurons.