Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38), known for its role in migraine pathogenesis, has been identified as a novel drug target. Given the clinical parallels between post-traumatic headache (PTH) and migraine, we explored the possible role of PACAP-38 in the pathogenesis of PTH. To this end, we conducted a randomized, double-blind, placebo-controlled, 2-way crossover trial involving adult participants diagnosed with persistent PTH resulting from mild traumatic brain injury. Participants were randomly assigned to receive a 20-minute continuous intravenous infusion of either PACAP-38 (10 pmol/kg/min) or placebo (isotonic saline) on two separate experimental days, with a 1-week wash-out period in between. The primary outcome was the difference in incidence of migraine-like headache between PACAP-38 and placebo during a 12-hour observational period post-infusion. The secondary outcome was the difference in the area under the curve (AUC) for baseline-corrected median headache intensity scores during the same 12-hour observational period. Of 49 individuals assessed for eligibility, 21 were enrolled and completed the trial. The participants had a mean age of 35.2 years, and 16 (76%) were women. Most of them (19 [90%] of 21) had a migraine-like phenotype. During the 12-hour observational period, 20 (95%) of 21 participants developed migraine-like headache after intravenous infusion of PACAP-38, compared with 2 (10%) participants after placebo (P < 0.001). Furthermore, the baseline-corrected AUC values for median headache intensity scores during the 12-hour observational period was higher after PACAP-38 than placebo (P < 0.001). These compelling results demonstrate that PACAP-38 is potent inducer of migraine-like headache in people with persistent PTH. Thus, targeting PACAP-38 signalling might be a promising avenue for the treatment of PTH.