Recent studies involving human and animal models have identified that saffron helps in the improvement of depression. Antidepressants are known to function in part by increasing brain serotonin, norepinephrine and dopamine concentrations. Therefore, to identify the cellular and molecular mechanism(s) underlying this property of saffron, we measured changes in rat brain dopamine, serotonin, norepinephrine and glutamate concentrations after administration of varying doses of an aqueous extract of saffron stigma. Male Wistar rats (250 ± 30 g) were administered a single dose of saffron extract (5, 25, 50, 100, 150, and 250 mg/kg, i.p.), fluoxetine (10 mg/kg, i.p.), and/or desipramine (50 mg/kg, i.p.) and were sacrificed 30 min later. Brains were removed, homogenized, and centrifuged at 4˚C. The supernatant was used for subsequent neurotransmitter detection by ELISA. Our results indicated that the aqueous extract of saffron (50, 100, 150 and 250 mg/kg, i.p.) increased brain dopamine concentration in a dose-dependent manner compared with saline. In addition, the brain glutamate concentration increased in response to the highest dose of the extract (250 mg/kg, i.p.). Interestingly, the extract had no effect on brain serotonin or norepinephrine concentration. Our findings show that the aqueous extract of saffron contains an active component that can trigger production of important neurotransmitters in brain, namely, dopamine and glutamate. In addition, these results provide a cellular basis for reports concerning the antidepressant properties of saffron extract in humans and animals.