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Substantial evidence points to melatonin as playing a role in the regulation of circadian rhythms, sleep, and headache disorders. The objective of the study was to assess 6-sulphatoxymelatonin (aMT6s) levels in a large consecutive series of patients with migraine, comparing with controls. A total of 220 subjects were evaluated—146 had migraine and 74 were control subjects. Urinary samples were collected into the same plastic container since 8:00 p.m. to 8:00 a.m. of the next day (12-h period) and aMT6s was measured with quantitative ELISA technique. Among patients with migraine, 53% presented pain on the day of the urine samples collection. Their urinary aMT6s concentration was significantly lower than in the urine of patients without pain [14.0 ± 7.3 vs. 49.4 ± 19.0; t(143) = −15.1; 95% CI = −40.0 to −30.8; P < 0.001]. There was no significant difference in the aMT6s concentration of patients with migraine without pain on the day of their urine samples collection and controls [49.4 ± 19.0 vs. 42.5 ± 27.9; t(140) = 1.7; 95% CI = −1.2 to 14.8; P = 0.094]. To our knowledge, this is the first study to demonstrate reduction in melatonin levels during attacks in episodic and chronic migraine.