Glycosaminoglycans (GAGs) contribute to the cellular uptake of cationic cell‐penetrating peptides (CPPs). However, molecular details about the contributions of GAGs in CPP internalization remain unclear. In this study, we examined the cellular uptake mechanism of the arginine‐rich CPP pituitary adenylate‐cyclase‐activating polypeptide (PACAP). We observed that the uptake efficacy of PACAP is dependent on the expression of cell surface GAGs. As the binding of PACAP to sulfated GAGs induced a random coil‐to‐α‐helix conformational conversion, we investigated the role of the helical formation in PACAP internalization. Whereas this secondary structure was not crucial for efficient internalization in GAGs‐deficient cells, PACAP α‐helix was essential for GAGs‐dependent uptake.