ObjectiveTemporal lobe epilepsy (TLE) is characterized by recurrent seizures generated in the limbic system, particularly in the hippocampus. In TLE, recurrent mossy fiber sprouting from dentate gyrus granule cells (DGCs) crea an aberrant epileptogenic network between DGCs which operates via ectopically expressed GluK2/GluK5‐containing kainate receptors (KARs). TLE patients are often resistant to anti‐seizure medications and suffer significant comorbidities; hence, there is an urgent need for novel therapies. Previously, we have shown that GluK2 knockout mice are protected from seizures. This study aims at providing evidence that downregulating KARs in the hippocampus using gene therapy reduces chronic epileptic discharges in TLE.MethodsWe combined molecular biology and electrophysiology in rodent models of TLE and in hippocampal slices surgically resected from patients with drug‐resistant TLE.ResultsHere, we confirmed the translational potential of KAR suppression using a non‐selective KAR antagonist that markedly attenuated interictal‐like epileptiform discharges (IEDs) in TLE patient‐derived hippocampal slices. An adeno‐associated virus (AAV) serotype‐9 vector expressing anti‐grik2 miRNA was engineered to specifically downregulate GluK2 expression. Direct delivery of AAV9‐anti grik2 miRNA into the hippocampus of TLE mice led to a marked reduction in seizure activity. Transduction of TLE patient hippocampal slices reduced levels of GluK2 protein and, most importantly, significantly reduced IEDs.InterpretationOur gene silencing strategy to knock down aberrant GluK2 expression demonstrates inhibition of chronic seizure in a mouse TLE model and IEDs in cultured slices derived from TLE patients. These results provide proof‐of‐concept for a gene therapy approach targeting GluK2 KARs for drug‐resistant TLE patients. ANN NEUROL 2023;94:745–761