Vitamin D deficiency (VDD) is prevalent among HIV-infected individuals. Vitamin D has been associated with renal and cardiovascular diseases because of its effects on oxidative stress, lipid metabolism and renin-angiotensin-aldosterone system (RAAS). Tenofovir disoproxil fumarate (TDF), a widely used component of antiretroviral regimens for HIV treatment, can induce renal injury. The aim of this study was to investigate the effects of VDD on TDF-induced nephrotoxicity. Wistar rats were divided into four groups: control, receiving a standard diet for 60 days; VDD, receiving a vitamin D-free diet for 60 days; TDF, receiving a standard diet for 60 days with the addition of TDF (50 mg/kg food) for the last 30 days; and VDD+TDF receiving a vitamin D-free diet for 60 days with the addition of TDF for the last 30 days. TDF led to impaired renal function, hyperphosphaturia, hypophosphatemia, hypertension and increased renal vascular resistance due to downregulation of the sodium-phosphorus cotransporter and upregulation of angiotensin II and AT1 receptor. TDF also increased oxidative stress, as evidenced by higher TBARS and lower GSH levels, and induced dyslipidemia. Association of TDF and VDD aggravated renovascular effects and TDF-induced nephrotoxicity due to changes in the redox state and involvement of RAAS.