Experimental data have demonstrated a role for S100B protein through the release of proinflammatory cytokines, following trigeminal nerve activation, implicated in the pathology of migraine. We investigated serum levels of S100B protein, as a peripheral glial biomarker, in patients with migraine. In total, 49 migraineurs and 35 age- and gender-matched controls were enrolled in this prospective clinical study. The migraine diagnosis was made according to the International Classification of Headache Disorders II diagnostic criteria. Serum samples were obtained for the measurement of S100B levels from all participants and were analyzed using commercial enzyme-linked immunosorbent assay kits. Serum S100B levels were significantly lower in migraineurs than controls (p < 0.001). S100B levels did not significantly differ in migraineurs with or without aura (p > 0.05). In addition, there was no correlation between serum S100B levels and headache characteristics, including attack severity, frequency and duration, and disease duration (p > 0.05). These findings suggest that serum S100B levels were significantly decreased in migraine patients, but further research is needed to ascertain the contribution of S100B in the clinical evaluation of migraine.