Coumarin is a compound known to be present in a wide variety of plants, microorganisms and animal species. Most of its effects were studied in organs and systems other than the central nervous system. The present work evaluated the effect of coumarin administration on the levels of gamma-aminobutyric acid (GABA), glutamate (GLU), glycine (GLY) and taurine (TAU) in the prefrontal cortex and hippocampus of mice. Male Swiss mice were treated with distilled water (controls), coumarin (20 or 40 mg/kg, i.p.) or diazepam (1 mg/kg, i.p.). Results showed that in the prefrontal cortex, coumarin at the lowest dose increased the levels of GLU and TAU, while GABA increased with both doses studied and GLY had its levels increased only at the dose of 40 mg/kg. Diazepam (DZP) increased the levels of GABA and TAU and decreased the levels of GLU and GLY in this area. In the hippocampus, only glutamate had its levels decreased after coumarin treatment, while diazepam increased the levels of GABA and TAU and decreased the levels of GLU in this brain region. We concluded that coumarin stimulates the release of endogenous amino acids, increasing the levels of inhibitory and excitatory amino acids in the prefrontal cortex, and decreasing glutamate levels in the hippocampus. Together, these results are of interest, considering that some neurodegenerative diseases and seizures are related to the imbalance of the amino acid levels in the CNS suggesting a perspective of a therapeutic use of coumarins in these disorders.