The axon initial segment (AIS) is located at the proximal axon, which is responsible for information output by generating action potentials. Recent studies have shown that the structure of AIS can be altered based on the input to the neuronal circuit, and this can impact neural activity. We previously observed that AIS length was altered in the cortical regions of both mice and rats with attention-deficit hyperactivity disorder (ADHD)-like behavior. These findings indicate that the abnormality of neural activity due to the alteration of AIS lengths is associated with ADHD. However, the link between AIS length changes and ADHD remains unclear. To investigate this further, we examined whether pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient (PACAP–/–) mice, which display ADHD-like behavior show AIS length alteration. Further, we also examined the effects of atomoxetine, a drug for ADHD on the AIS length in PACAP–/– mice. We found that PACAP–/– mice exhibited a longer AIS length compared with wild-type mice. In addition, repeated treatments with atomoxetine improved AIS abnormality along with hyperactivity in PACAP–/– mice. These results suggest that AIS abnormality is one of the phenotypes of ADHD and improving AIS abnormalities could be a novel drug target for ADHD pathophysiology.