Introduction Administration of ATP-sensitive potassium channel opener levcromakalim triggers headache in healthy volunteers and migraine attacks in migraine patients. Here, we investigated the effect of ATP-sensitive potassium channel blocker glibenclamide on levcromakalim-induced headache in healthy volunteers. Methods In a randomized, double-blind, placebo-controlled, three-way cross-over study, 15 healthy volunteers aged 18–40 years were randomly allocated to receive glibenclamide and levcromakalim (day 1), glibenclamide and placebo (day 2), and placebo and placebo (day 3) on three different days separated by at least 1 week. The primary endpoints were the difference in incidence of headache and the difference in area under the curve for headache intensity scores (0–12 hours) between the days. Results Fifteen healthy volunteers completed the 3 days of the study. More participants (12/15, 80%) developed headache on the glibenclamide-levcromakalim day compared to the glibenclamide-placebo day (5/15, 33%) ( p = 0.01; mean difference 47%; 95% confidence interval 18–75%) and compared to the placebo-placebo day (1/15, 7%) ( p = 0.001; mean difference 73%; 95% confidence interval 48–99%). We found no difference in headache incidence between glibenclamide-placebo day and placebo-placebo day ( p = 0.12; mean difference 27%; 95% confidence interval 1.3–52%). The area under the curve for headache intensity was significantly larger on the glibenclamide-levcromakalim day compared to the glibenclamide-placebo day ( p = 0.003); and compared to the placebo-placebo day ( p = 0.001). We found no difference in the area under the curve between the glibenclamide-placebo day compared to the placebo-placebo day ( p = 0.07). The median time to onset for headache after levcromakalim infusion with glibenclamide pretreatment was delayed (180 min) compared to levcromakalim without pretreatment (30 min) from a previously published study. Conclusion Glibenclamide administration did not cause headache, and glibenclamide pretreatment did not prevent levcromakalim-induced headache. However, glibenclamide delayed the onset of levcromakalim-induced headache. More selective blockers are needed to further elucidate the role of the ATP-sensitive potassium channel in headache initiation. Trial Registration: ClinicalTrials.gov NCT03886922.