Drug-induced kidney injury in medicinal compound development accounts for over 20% of clinical trial failures and involves damage to different nephron segments, mostly the proximal tubule. Yet, currently applied cell models fail to reliably predict nephrotoxicity; neither are such models easy to establish. Here, we developed a novel three-dimensional (3D) nephrotoxicity platform on the basis of decellularized rat kidney scaffolds (DS) recellularized with conditionally immortalized human renal proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1). A 5-day SDS-based decellularization protocol was used to generate DS, of which 100-μm slices were cut and used for cell seeding. After 8 days of culturing, recellularized scaffolds (RS) demonstrated 3D-tubule formation along with tubular epithelial characteristics, including drug transporter function. Exposure of RS to cisplatin (CDDP), tenofovir (TFV), or cyclosporin A (CsA) as prototypical nephrotoxic drugs revealed concentration-dependent reduction in cell viability, as assessed by PrestoBlue and Live/Dead staining assays. This was most probably attributable to specific uptake of CDDP by the organic cation transporter 2 (OCT2), TFV through organic anion transporter 1 (OAT1), and CsA competing for P-glycoprotein-mediated efflux. Compared with 2D cultures, RS showed an increased sensitivity to cisplatin and tenofovir toxicity after 24-hour exposure (9 and 2.2 fold, respectively). In conclusion, we developed a physiologically relevant 3D nephrotoxicity screening platform that could be a novel tool in drug development.