Multidrug and toxin extrusion (MATE) 1 and MATE2-K, H(+)/organic cation antiporters, are located at the brush-border membrane of renal proximal tubules. The present study aimed to clarify the role of MATE transporters in tubular secretion of varenicline. Varenicline at a dose of 5 mg/kg was administered to wild-type and Mate1-knockout mice via the jugular vein, and its uptake was measured by high-performance liquid chromatography. The renal secretory clearance of and systemic exposure to varenicline were significantly decreased (54.6%, p < 0.05) and increased (116%, p < 0.05) respectively, by the genetic disruption of Mate1 in mice. Uptake of varenicline and [(14)C]tetraethylammonium (TEA) was examined in HEK293 cells transiently expressing the human (h) MATE1, hMATE2-K, mouse (m) MATE1, and hOCT2 basolateral organic cation transporter. [(14)C]TEA uptake in HEK293 cells expressing MATE transporters and hOCT2 was decreased in the presence of varenicline. The calculated IC(50) values for hMATE1, hMATE2-K, mMATE1, and hOCT2 were 62.2 ± 6.5, 122.3 ± 67.6, 255.0 ± 37.9, and 1,003.9 ± 135.8 (µM; mean ± S.E. for three separate experiments), respectively. Varenicline uptake was significantly increased in HEK293 cells expressing mMATE1, hMATE1, or hMATE2-K cDNA as well as hOCT2 compared to empty vector-transfected cells. In conclusion, renal MATE transporters were found to be responsible for renal tubular secretion of varenicline.