Glycine receptor (GlyR) α3 is involved in vision, and processing of acoustic and nociceptive signals, and RNA editing of GLRA3 transcripts was associated with hippocampal pathophysiology of mesial temporal lobe epilepsy (TLE). However, neither the role of GlyR α3 splicing in hippocampal neurons nor the expression of splice variants have yet been elucidated. We report here that the long (L) splice variant of GlyR α3 predominates in the brain of rodents. Cellular analysis using primary hippocampal neurons and hippocampus cryosections revealed preferential association of synaptic α3L clusters with glutamatergic nerve endings in strata granulare and pyramidale. In primary hippocampal neurons GlyR α3L clusters also preferred glutamatergic nerve endings while α3K was mainly in a diffuse state. Co‐expression of GlyR β subunit with α3L or α3K produced heteromeric receptor clusters and favoured their association with GABAergic terminals. However, heteromeric α3L was still more efficient than heteromeric α3K in associating with glutamatergic nerve endings. To give physiological relevance to these results we have finally analysed GlyR α3 splicing in human hippocampus obtained from patients with intractable TLE. As up‐regulation of α3K occurred at the expense of α3L in TLE patients with a severe course of disease and a high degree of hippocampal damage, our results again involve post‐transcriptional processing of GLRA3 transcripts in the pathophysiology of TLE.