Pituitary adenylate cyclase-activating polypeptide (PACAP) has emerged as an important signaling peptide in migraine pathogenesis. Recently, we have shown that the less-abundant PACAP isoform, PACAP27, induced migraine and headache in patients equipotently to PACAP38. The present study examined the effect of PACAP27 on cerebral hemodynamics in healthy volunteers using high resolution magnetic resonance angiography (MRA). Eighteen healthy volunteers received infusion of PACAP27 (10 pmol/kg/min) or placebo over 20 min and were scanned repeatedly in fixed intervals for 5 h in a double-blind, randomized, placebo-controlled study. The circumference of extra-intracerebral arteries was measured and compared with PACAP38 data. We found significant dilation of middle meningeal artery (MMA) (p = 0.019), superficial temporal artery (p = 0.001) and external carotid artery (p = 0.039) after PACAP27 infusion compared to placebo. Whereas the middle cerebral artery (MCA) (p = 0.011) and internal carotid artery (ICA) (p_ICAcervical = 0.015, p_ICAcerebral = 0.019) were constricted. No effects on basilar artery (p = 0.708) and cavernous portion of ICA were found. Post hoc analyses revealed significant larger area under the curve for MMA after PACAP38 compared to PACAP27 (p = 0.033). We also found that PACAP27 induced headache in nine out of twelve (75%) volunteers and one (17%) after placebo. In conclusion, PACAP27 induced headache and dilated extracerebral arteries (>5 h) and slightly constricted MCA in healthy volunteers. Post hoc analysis of PACAP38 data compared with PACAP27 showed that PACAP isoforms dilates MMA with significantly different magnitude.