Pituitary adenylate cyclase activating polypeptide (PACAP) has several different actions in the nervous system. Numerous studies have shown its neuroprotective effects both in vitro and in vivo. Previously, it has been demonstrated that PACAP reduces brain damage in rat models of global and focal cerebral ischemia. Based on the protective effects of PACAP in cerebral ischemia and the presence of common pathogenic mechanisms in cerebral ischemia and traumatic brain injury (TBI), the aim of the present study was to investigate the possible protective effect of PACAP administered 30 min or 1 h postinjury in a rat model of diffuse axonal injury. Adult Wistar male rats were subjected to impact acceleration, and PACAP was administered intracerebroventricularly 30 min (n = 4), and 1 h after the injury (n = 5). Control animals received the same volume of vehicle at both time-points (n = 5). Two hours after the injury, brains were processed for immunohistochemical localization of damaged axonal profiles displaying either beta-amyloid precursor protein (beta-APP) or RMO-14 immunoreactivity, both considered markers of specific features of traumatic axonal injury. Our results show that treatment with PACAP (100 microg) 30 min or 1 h after the induction of TBI resulted in a significant reduction of the density of beta-APP-immunopositive axon profiles in the corticospinal tract (CSpT). There was no significant difference between the density of beta-APP-immunopositive axons in the medial longitudinal fascicle (MLF). PACAP treatment did not result in significantly different number of RMO-14-immunopositive axonal profiles in either brain areas 2 hours post-injury compared to normal animals. While the results of this study highlighted the complexity of the pathogenesis and manifestation of diffuse axonal injury, they also indicate that PACAP should be considered a potential therapeutic agent in TBI.