A large amount of literature has indicated that excitatory synaptic transmission plays a crucial role in epilepsy, but the detailed pathogenesis still needs to be clarified. In the present study, we used samples from patients with temporal lobe epilepsy, pentylenetetrazole-kindled mice, and Mg2+ -free-induced epileptic cultured hippocampal neurons to detect the expression pattern of STK24. Then, the whole-cell recording was carried out after STK24 overexpression in the Mg2+ -free-induced epileptic cultured hippocampal neurons. In addition, coimmunoprecipitation was performed to detect the association between endogenous STK24 and main subunits of NMDARs and AMPARs in the hippocampus of PTZ-kindled mice. Here, we reported that STK24 was specifically located in epileptic neurons of human and pentylenetetrazole-kindled mice. Meanwhile, the expression of STK24 was significantly down-regulated in these samples which are mentioned above. Besides, we found that the amplitude of miniature excitatory postsynaptic currents was increased in STK24 overexpressed epileptic hippocampal cultured neurons, which means the excitatory synaptic transmission was changed. Moreover, the coimmunoprecipitation, which further supported the previous experiment, indicated an association between STK24 and the subunits of the NMDA receptor. These findings expand our understanding of how STK24 involved in the excitatory synaptic transmission in epilepsy and lay a foundation for exploring the possibility of STK24 as a drug target. © 2020 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.