Objectives The purpose of this study is to verify the phenytoin-resistant mesial temporal lobe epilepsy (MTLE) induced by Li-pilocarpine and screened by antiepilepsy drug (AEDs). Methods The rats with MTLE were induced by Li-pilocarpine, which were screened by effect of phenytoin treatment monitored by vedio-EEG. The living microdialysis technology was used for verification of drug concentration in brain of drug-resistant and drug-responsive rat model, and the P-glycoprotein expression was detected by immunohistochemical method. Results Sixteen rats with chronic MTLE were successfully induced in total 30 rats, among which, 6 drug-resistant rats with MTLE were screened. The brain/plasma ratio of area under the curve in drug-resistant rats was significantly lower than that of drug-responsive rats (0.15±0.03 vs. 0.28±0.05, P<0.05). In addition, the P-glycoprotein expression in brain of drug-responsive rats was obviously higher than that of drug-responsive rats (P<0.05). Conclusions The low concentration of phenytoin in drug-resistant rat model with MTLE was verified that might be related to the over-expressed P-glycoprotein in brain.