Temporal lobe epilepsy (TLE), the most common form of DRE, is associated with enhanced glutamate receptor-mediated hyperexcitability in the temporal lobe structures. The mechanism of regulation of aberrant glutamatergic activity in TLE is still elusive. Sema 3F regulates the AMPA receptor surface expression in post-synapse. In this study, we investigated the contribution of semaphorin3F (SEMA 3F) to glutamatergic synaptic activity in TLE rats' hippocampus and anterior temporal lobe (ATL). synaptic transmission in the hippocampus and ATL. Golgi-Cox staining was conducted to visualize alterations in neuronal morphology, including their soma, axons, dendrites, and spines. Increased glutamatergic activity was observed in the hippocampus and ATL samples from TLE rats, with a more pronounced increase in the hippocampus. At the mRNA level, significant upregulation of SEMA3F and AMPA receptor subunit GLUR4 was observed in the hippocampus and ATL of the TLE. We observed a significant reduction in the seizure frequency in TLE rats injected with siRNA for SEMA 3F. However, no changes in SEMA3F mRNA levels or AMPA receptor GLUR4 expression were observed in the hippocampus and ATL in TLE rats injected with siRNA for SEMA 3F. However, protein expression of SEMA3F and GLUR4 was decreased in the siRNA-injected in these rats. Furthermore, in the hippocampal pyramidal neurons of TLE rats, we observed altered lengths of apical and basal dendrites, changes in spine density, and modifications in soma architecture. This study provides the first direct evidence that altered levels of SEMA3F could be contributing to enhanced AMPA receptor-mediated glutamatergic synaptic transmission in TLE.