Ethambutol is a potent antimycobacterial agent used in tuberculosis (TB) treatment. Renal elimination is the major route and recently reported as a substrate of organic cation transporters. We previously reported that genetic polymorphism in OCT transporters has decreased metformin clearance. Therefore, we aimed to evaluate effect of OCT2 variants on ethambutol transport in vitro and prediction of pharmacokinetics using physiologically based pharmacokinetic modeling. The transport experiments were conducted using human embryonic kidney cells stably transfected with human OCT2 transporter wild type and variants (T199I, T201M and A270S) in vitro. A full PBPK model for ethambutol was developed following permeability limited mechanistic kidney model (MechKim) in the SIMCYP simulator (Ver 15.1) using in vitro data. A 25mg/kg single dose for the healthy subjects in the simulator was used to build the model. It was then optimized and verified using clinical pharmacokinetic data. The verified PBPK model was then used to predict the effect of OCT2 genetic variants on ethambutol pharmacokinetics in healthy subjects in the SIMCYP population database. The ethambutol uptake was saturable and active for OCT2. The genetic variants of OCT2-T199I (1% in KOR), -T201M (2% in KOR) and -A270S (14% in OR) were reduced 3.2-fold, 7-fold, and 1.7-fold ethambutol transport compared with wild-type, respectively. The predicted pharmacokinetics showed the unchanged amount of ethambutol excretion in urine and the total amount in kidney cells were 2-5 fold difference between the wild and mutant groups. The AUC ratio were 1.42, 1.40 and 1.36 fold higher and CL were 30.2, 29.3 and 28.7 % lower for having T199I, T201M and A270S subjects compared to wild type. This in vitro to in vivo extrapolation study showed genetic polymorphism in OCT2 transporter affected on ethambutol pharmacokinetics which may explain interindividual response.