Ethanol withdrawal commonly leads to anxiety-related disorder, a central factor toward negative reinforcement leading to relapse. The lateral habenula (LHb), an epithalamic nucleus, has emerged to be critical for both reward and aversion processing. Recent studies have also implicated the hyperactivity of LHb, adding to the emergence of negative emotional states during withdrawal from addictive drugs. Herein, we have studied the effects of glutamate transporter inhibitor (PDC), GluN2B-containing NMDAR antagonist (Ro25-6981), and intracellular calcium chelator (BAPTA-AM) injection in LHb on ethanol withdrawal symptoms. We found that ethanol 4 g/kg 20 % w/v intragastric (i.g.) for 10 days followed by 24 h of withdrawal showed a significant increase in somatic signs characterized by vocalization, shaking, and scratching. It also increased locomotor activity and anxiety-like behavior, collectively showing expression of ethanol withdrawal symptoms. The intra-LHb administration of PDC (0.5 ng) worsened the effect of ethanol withdrawal, whereas Ro25-6981 (2 and 4 ng) and BAPTA-AM (6.5 and 13 ng) significantly reversed ethanol withdrawal-induced behavior evident by a decrease in somatic signs, locomotor activity, and anxiety-like behavior. Further, pretreatment of Ro25-6981 and BAPTA-AM reduced the neuronal loss, whereas PDC increased it compared to the vehicle-treated group, as evidenced by NeuN staining. Altogether, our results suggest that increased glutamate, GluN2B activation, and likely calcium increase indicative of glutamate excitotoxicity-induced neuronal loss in LHb possibly endorse the emergence of ethanol withdrawal symptoms, while their inhibition might help in alleviating the ethanol withdrawal symptoms.