Chronic exposure to Cd²⁺ causes renal proximal tubular (PT) damage. Cd²⁺ reaches the PT mainly as cadmium-metallothionein 1 (CdMT-1) complexes which are filtered at the glomerulus and then internalized in part via endocytosis mediated by megalin and cubulin. Subsequently, Cd²⁺ is thought to be released into the cytosol to activate cell death pathways. The proton-coupled divalent metal transporter DMT1 also transports Cd²⁺ and is expressed exclusively in endosomes/lysosomes in rat PT cells. Using vector-based RNA interference with short-hairpin siRNAs (shRNAs) to downregulate DMT1 in the rat renal PT cell line WKPT-0293 Cl.2, we tested the hypothesis that endosomal/lysosomal DMT1 is involved in CdMT-1 nephrotoxicity. One out of 5 shRNAs tested (sh3) significantly reduced expression of DMT1 protein detected by immunoblotting as well as DMT1 mRNA as determined by RT-PCR by 45.1 ± 9.6% and 36.9 ± 14.4% (n=5-6), respectively. Similarly, sh3 reduced perinuclear DMT1 immunostaining in transfected cells. Consistent with the assumed role of DMT1 in CdMT-1 cytotoxicity, sh3, but not the empty vector or sh5, significantly attenuated cell death induced by a 24 h exposure to 14.3 µM CdMT-1 by 35.6 ± 4.2% (n = 13). In contrast, neither fluorescently-labeled MT-1 uptake nor free Cd²⁺ toxicity were altered by the effective DMT1 shRNA (sh3), indicating that cellular uptake of metal-MT-1 complexes and Cd²⁺-induced cell death signaling are not affected by DMT1 knockdown. Thus, we conclude that endosomal/lysosomal DMT1 plays a role in renal PT CdMT-1 toxicity.