Pituitary adenylate cyclase-activating polypeptide (PACAP) affects rodents' stress-related behaviors, such as anxiety-like behavior or fear conditioning. However, previous studies have investigated the effect of intracerebroventricular, but not hippocampal, injection of this PAC1R-selective antagonist (PACAP-6-38) on anxiety-like behavior. However, it has been reported that administration of PACAP-6-38 to the dorsal hippocampus reduces the fear response in a fear conditioning test. Therefore, this study aimed to determine whether the effect of dorsal hippocampal PACAP-6-38 injection in Sprague-Dawley rats can affect anxiety-like behavior assessed by an elevated plus-maze test. As a result, rats treated with PACAP-6-38 spent longer time in open arms than those with saline, suggesting that this drug has an anxiolytic effect. In conclusion, the role of PACAP in the dorsal hippocampus can promote anxiety-like behavior.