A well-balanced intracellular iron trafficking in glial cells plays a role in homeostatic processes. Elevated intracellular iron triggers oxidative stress and cell damage in many neurological disorders, including migraine. This study aimed to investigate the effects of glyceryl trinitrate (GTN), on cellular iron homeostasis, matrixmetalloproteinase (MMP)-9, and calcitonin gene related peptide (CGRP) receptor (CRLR/CGRPR1) production in microglia, astrocyte, and meningeal cell cultures. Primary glial and meningeal cells in culture were exposed to GTN for 24 h. Messenger RNA expression was assessed using qPCR. Iron accumulation was visualized via modified Perl's histochemistry. MMP-9 levels in cell culture supernatants were measured using ELISA. Ferritin and CRLR/CGRPR1 proteins were visualized via immunofluorescence staining. Nitric oxide production increased significantly with GTN in meningeal and glial cells. GTN significantly increased the expression of the storage protein ferritin for all three cell types, but ferritin-L for meningeal cells and microglia. Iron trafficking associated with the efflux protein ferroportin and influx protein divalent metal transporter (DMT)1 was affected differently in all three cell types. MMP-9 expression was increased in astrocytes. GTN stimulation increased both CRLR/CGRPR1 expression, and immunostaining was apparent in microglia and meningeal cells. This study showed for the first time that GTN modulates intracellular iron trafficking regulated by storage and transport proteins expressed in meningeal cells and glia. CRLR/CGRPR1 expression might be related to altered iron homeostasis and they both may stimulate nociceptive pathways activated in migraine. These molecules expressed differently in glial and meningeal cells in response to GTN may bring not only new targets forward in treatment but also prevention in migraine.