Epilepsy is a common neurological disorder and glutamate excitotoxicity plays a key role in epileptic pathogenesis. Astrocytic glutamate transporter GLT-1 is responsible for preventing excitotoxicity via clearing extracellular accumulated glutamate. Previously, three variants (G82R, L85P, and P289R) in SLC1A2 (encoding GLT-1) have been clinically reported to be associated with epilepsy. However, the functional validation and underlying mechanism of these GLT-1 variants in epilepsy remain undetermined. In this study, we reported that these disease-linked mutants significantly decrease glutamate uptake, cell membrane expression of the glutamate transporter, and glutamate-elicited current. Additionally, we found that these variants may disturbed stromal-interacting molecule 1 (STIM1)/Orai1-mediated store-operated Ca²⁺ entry (SOCE) machinery in the endoplasmic reticulum (ER), in which GLT-1 may be a new partner of SOCE. Furthermore, knock-in mice with disease-associated variants showed a hyperactive phenotype accompanied by reduced glutamate transporter expression. Therefore, GLT-1 is a promising and reliable therapeutic target for epilepsy interventions.