Increasing evidences have shown that many pharmaceutical excipients are not pharmacologically inert but instead have effects on several transport function of uptake and efflux drug transporters. Herein, we investigated whether the excipients frequently used in many drug formulations affect transport function of organic cation transporters (OCTs) that are responsible for elimination of cationic drugs. Our finding revealed that solubilizing agents, Tweens, showed the most significant effect rbOCT1/2-mediated [3H]-MPP+ uptake in heterologous expressing cells. The haft inhibitory concentration (IC50) values of Tween 20, Tween 60, and Tween 80 for OCT1 were 85 ± 1.12, 50 ± 1.26, 106.00 ± 1.20 μg/ml, respectively, while the IC50 values for OCT2 were 295 ± 1.48, 42 ± 1.15, 185 ± 1.20 μg/ml, respectively. The inhibitory effect of Tween 20, Tween 60 and Tween 80 on OCT2-mediated [3H]-MPP+ uptake in the human renal proximal tubular cells (RPTEC/TERT1 cells) was found and the IC50 values was similar to heterologous OCT2 expressing cells. Interestingly, Tween 20, Tween 60 and Tween 80 exhibited less inhibitory effect on OCT1 functions in HepG2 cells expressing OCT1 compared with heterologous OCT1 expressing cells. In addition, clearance of [3H]-MPP+ was reduced in mice receiving Tween 20 compared with vehicle. The present study demonstrates that Tweens (solubilizing excipients) can inhibit the transport functions of OCT1 and OCT2, which play crucial roles for the pharmacokinetics, drug-drug interactions and tissue deposition of many cationic drugs.