Background and Purpose Glutamate receptor mediated enhanced excitatory neurotransmission is typically associated with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS). Kynurenic acid (KYNA) and quinolinic acid (QUIN) are two important tryptophan-kynurenine pathway (KP) metabolites that modulate glutamate receptor activity. This study was designed to test the hypothesis that alteration in metabolism of KP metabolites in the hippocampus of patients with MTLE-HS contributes to abnormal glutamatergic transmission. Experimental Approach TKP metabolites level were determined using HPLC and LC-MS/MS in the hippocampal samples of patients with MTLE-HS compared to autopsy and non-seizure control samples. mRNA and protein expression of TKP enzymes were determined by qPCR and western blot. Spontaneous glutamatergic activities were recorded from pyramidal neurons in presence of kynurenine (KYN) and KYNA using whole cell patch clamp. Key Results We observed significantly reduced KYNA and elevated QUIN levels in the hippocampal samples, while KYN level remains unaltered. Spontaneous glutamatergic activity in the hippocampal samples was higher compared to that in non-seizure controls. Treatment with kynurenine inhibited the glutamatergic activity in non-seizure control samples but not in case of the hippocampal samples. However, exogenously applied KYNA inhibited glutamatergic activity in both non-seizure control and hippocampal samples. We also observed reduced levels of enzyme kynurenine aminotransferase II and its co-factor pyridoxal phosphate in the hippocampal samples. Conclusion Our findings indicate that altered metabolism of TKP metabolites in hippocampus could contribute to hyperglutamatergic tone in patients with MTLE-HS.