Alcohol withdrawal syndrome (AWS) is a medical emergency situation which appears after abrupt cessation of ethanol intake. Decreased GABA-A function and increased glutamate function are known to exist in the AWS. However, the involvement of glutamate transporters in the context of AWS requires further investigation. In this study, we used a model of ethanol withdrawal involving abrupt cessation of binge ethanol administration (4 g/kg/gavage three times a day for three days) using male alcohol-preferring (P) rats. After 48 h of withdrawal, P rats were re-exposed to voluntary ethanol intake. The amount of ethanol consumed was measured during post-withdrawal phase. In addition, the expression of GLT-1, GLAST and xCT were determined in both medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). We also measured glutamine synthetase (GS) activity, and the tissue content of glutamate, glutamine, dopamine and serotonin in both mPFC and NAc. We found that binge ethanol withdrawal escalated post-withdrawal ethanol intake, which was associated with downregulation of GLT-1 expression in both mPFC and NAc. The expression of GLAST and xCT were unchanged in the ethanol-withdrawal (EW) group compared to control group. Tissue content of glutamate was significantly lower in both mPFC and NAc, whereas tissue content of glutamine was higher in mPFC but unchanged in NAc in the EW group compared to control group. The GS activity was unchanged in both mPFC and NAc. The tissue content of DA was significantly lower in both mPFC and NAc, whereas tissue content of serotonin was unchanged in both mPFC and NAc. These findings provide important information of the critical role of GLT-1 in context of AWS.