Abstract: Growing evidence suggests that esculetin, a 5-lipoxygenase inhibitor, has pharmacotherapeutic potential due to various pharmacological properties, such as potent anti-inflammatory, anti-nociceptive, and GABA(A) receptor partial agonist activities. However, the effects of this promising agent on migraine remain unexplored. This study therefore examined the impact of esculetin on the relevant mechanisms in migraine-like conditions in rats. The systemic effects of esculetin at three distinct doses (5, 10, and 20 mg/kg) were tested in a nitroglycerin-induced migraine model using in vivo experimental sets. The direct action of esculetin on the release of calcitonin gene-related peptide (CGRP) from critical structures of the trigeminovascular system (trigeminal ganglion, nucleus trigeminalis, and meningeal afferents) was also tested in ex vivo experimental sets. Sumatriptan was used as a positive control in both sets of experiments. The in vivo results showed that esculetin reduced NTG-induced mechanical hyperalgesia and decreased trigeminal CGRP and c-Fos levels. It also lowered degranulation and meningeal mast cell numbers. The ex vivo results revealed that esculetin reduced NTG-stimulated CGRP release from trigeminovascular explants, with the exception of meningeal explants. Sumatriptan reversed the NTG-induced changes in both experimental sets. Our findings suggest that esculetin exhibits anti-nociceptive activities in experimental migraine conditions, alleviating trigeminovascular CGRP concentrations and the degranulation of meningeal mast cells. Esculetin may thus represent a therapeutic option for relieving migraine headaches, although further research is needed to confirm this.