P-glycoprotein (P-gp), encoded by the MDR1 (multidrug resistance 1) gene, involves in the efflux of multiple compounds, such as certain antiepileptic drugs. The aim of this research was to observe the impacts of MDR1 (C3435T) variant on the efflux of phenytoin, carbamazepine, valproate, and phenobarbital in vitro. Stable recombinant LLC-PK1 cell systems transfected with MDR13435C (wild-type allele) and MDR13435T (variant allele) were constructed. The influences of MDR1 (C3435T) variant on the sensitivity, intracellular accumulation, and transepithelial permeability of antiepileptic drugs were assessed. The recombinant MDR13435T cells showed higher resistance to carbamazepine compared with MDR13435C cells in the cytotoxicity assay (p < 0.01). The intracellular accumulation of carbamazepine was significantly decreased in cells transfecting with MDR13435T allele when compared with recombinant MDR13435C cells (p < 0.01). These results also indicate that the efflux activity of P-gp-mediated carbamazepine in recombinant MDR13435T cells was greatly increased compared with MDR13435C cells (p < 0.01), whereas the transport ability of P-gp-dependent phenobarbital in recombinant MDR13435T cells was significantly lower than MDR13435C cells (p < 0.01). However, the effects of MDR1 (C3435T) polymorphism on the resistance, intracellular accumulation, and efflux of phenytoin and valproate were not found in this study. MDR13435T variant allele might be more efficient to transport carbamazepine, whereas reduces the efflux activity of P-gp-mediated phenobarbital. Collectively, MDR1 (C3435T) polymorphism might impact the P-gp activity and antiepileptic agents efflux with drug specificity.