An increasing number of epilepsy patients are afflicted with drug-resistant temporal lobe epilepsy (TLE) and require alternative therapeutic approaches. High-affinity glycine receptors (haGlyRs) are functionally adapted to tonic inhibition due to their response to hippocampal ambient glycine, and their synthesis is activity-dependent. Therefore, in our study, we scanned TLE hippocampectomies for expression of haGlyRs and characterized the effects mediated by these receptors using primary hippocampal neurons. Increased haGlyR expression occurred in TLE hippocampi obtained from patients with a severe course of disease. Furthermore, in TLE patients, haGlyR and potassium chloride cotransporter 2 (KCC2) expressions were inversely regulated. To examine this potential causal relationship with respect to TLE histopathology, we established a hippocampal cell culture system utilising tonic inhibition mediated by haGlyRs in response to hippocam-pal ambient glycine and in the context of a high Cl equilibrium potential, as is the case in TLE hippocampal neurons. We showed that hypoactive neurons increase their ratio between glutamatergic and GABAergic synapses, reduce their dendrite length and finally undergo excitotoxicity. Pharmacological dissection of the underlying processes revealed ionotropic glutamate and TrkB receptors as critical mediators between neuronal hypoactivity and the emergence of these TLE-characteristic histopathological signs. Moreover, our results indicate a beneficial role for KCC2, because decreasing the Cl− equilibrium potential by KCC2 expression also rescued hypoactive hippocampal neurons. Thus, our data support a causal relationship between increased haGlyR expression and the emergence of histopathological TLE-characteristic signs, and they establish a pathophysiological role for neuronal hypoactivity in the context of a high Cl− equilibrium potential.