Tenofovir disoproxil fumarate (TDF), a nucleotide reverse transcriptase inhibitor, after conversion to TFV, is mainly eliminated by glomerular filtration and active tubular secretion. The major adverse effect of tenofovir is nephrotoxicity, however, the exact mechanism remains poorly understood. In this study, ABCC11 (MRP8) transporter, a member of ATP-21 binding cassette subfamily C11, which is abundant in proximal tubular cells, was demonstrated to efflux tenofovir. Real-time polymerase chain reaction (rt-PCR) and indirect immunofluorescence assays were used to determine MRP8 overexpression in a continuous Society for Microbiology. All Rights Reserved. Tenofovir accumulations were assessed by cytotoxicity, cellular transport, and vesicular uptake assays. Substrate specificity was confirmed using MK-571, an MRP- specific inhibitor, and methotrexate which served as a known substrate. Intracellular and intravesicular concentrations of tenofovir were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The 50% cytotoxic concentrations (CC50) of TDF in MRP8-overexpressed cells was 4.78 times higher when compared to that of parental cells. Transport assays also showed that the intracellular accumulation of tenofovir in MRP8-31 overexpressed cells was 55 times lower than that of the parental cells, and was partly reversed by MK-571. Similarly, the inside-out vesicular uptake assay demonstrated higher intravesicular concentration of tenofovir in MRP8-overexpressed vesicles than that of the Sf9 insect vesicles. These effects were effectively reversed by increasing concentrations of 35 specific inhibitor, MK-571. In conclusion, tenofovir is a new substrate of MRP8 transporter. An alteration in the activity of this efflux pump may increase the intracellular accumulation 37 of tenofovir in proximal renal tubular cells.