Renal magnesium (Mg²⁺) and sodium (Na⁺) loss are well-known side effects of cyclosporine (CsA) treatment in humans, but the underlying mechanisms still remain unclear. Recently, it was shown that epidermal growth factor (EGF) stimulates Mg²⁺ reabsorption in the distal convoluted tubule (DCT) via TRPM6 (Thébault S, Alexander RT, Tiel Groenestege WM, Hoenderop JG, Bindels RJ. J Am Soc Nephrol 20: 78–85, 2009). In the DCT, the final adjustment of renal sodium excretion is regulated by the thiazide-sensitive Na⁺-Cl⁻ cotransporter (NCC), which is activated by the renin-angiotensin-aldosterone system (RAAS). The aim of this study was to gain more insight into the molecular mechanisms of CsA-induced hypomagnesemia and hyponatremia. Therefore, the renal expression of TRPM6, TRPM7, EGF, EGF receptor, claudin-16, claudin-19, and the NCC, and the effect of the RAAS on NCC expression, were analyzed in vivo in a rat model of CsA nephrotoxicity. Also, the effect of EGF administration on these parameters was studied. CsA significantly decreased the renal expression of TRPM6, TRPM7, NCC, and EGF, but not that of claudin-16 and claudin-19. Serum aldosterone was significantly lower in CsA-treated rats. In control rats treated with EGF, an increased renal expression of TRPM6 together with a decreased fractional excretion of Mg²⁺ (FE Mg²⁺) was demonstrated. EGF did not show this beneficial effect on TRPM6 and FE Mg²⁺ in CsA-treated rats. These data suggest that CsA treatment affects Mg²⁺ homeostasis via the downregulation of TRPM6 in the DCT. Furthermore, CsA downregulates the NCC in the DCT, associated with an inactivation of the RAAS, resulting in renal sodium loss.