<p>Growing evidence suggests that esculetin, a 5-lipoxygenase inhibitor, has pharmacotherapeutic potential due to its various pharmacological properties, such as potent anti-inflammatory, anti-nociceptive, and γ-aminobutyric acid type A (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 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 (NTG)-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, trigeminal nucleus, 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 cellular Fos proto-oncogene (c-Fos) levels. It also decreased 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.</p>