OBJECTIVES: Investigating the effect of melatonin (MLT) on the pharmacokinetics and related neurotransmitter and amino acid metabolism of vigabatrin (VGB) in epileptic rats in vivo. METHODS: High performance liquid chromatography was used to examine the pharmacokinetics and tissue distribution of VGB after intragastric administration dosing (50，100，200) mg/kg singly or in combination with melatonin (20 mg/kg) in rats. The single-compartment model of first-order elimination was fitted with the nonlinear mixed-effect model of first-order estimation. Targeting metabolomics were used to measure and analyze the amino acid levels in the hippocampus of kainic acid (KA)-induced epileptic rats treated with VGB alone or coupled melatonin. RESULTS: Melatonin significantly alters the pharmacokinetics of VGB, primarily by lengthening the elimination t CONCLUSIONS: MLT affected the pharmacokinetics and tissue distribution of VGB in rats, prolonging its elimination time and improving the tissue distribution. Moreover, it might help VGB improve the imbalance of neurotransmitters and amino acids in the hippocampus of epileptic rats.