Acetaminophen (N-acetyl-p-aminophenol; APAP), a widely used analgesic and antipyretic drug, can cause life-threatening renal dysfunction. Factors which play a role in this toxicity are still ambiguous and no specific treatment was ascertained. The aim of this study was to investigate the potential protective role of silymarin to attenuate the nephrotoxicity induced by a single oral dose (3 g/kg) of APAP in rats. Four groups of Sprague-Dawley rats were used: control, silymarin, APAP and silymarin plus APAP-receiving animals. Interestingly, oral supplementation of silymarin (200 mg/kg/day) for nine days before APAP intoxication dramatically reduced APAP-induced nephrotoxicity as evidenced by measuring serum total protein, serum urea, creatinine clearance (Ccr) and urinary excretions of NAG (N-acetyl-β-D-glucosaminidase). Silymarin administration markedly prevented the generation of thiobarbituric acid reacting substances (TBARS) with substantial improvement in terms of reduced glutathione content (GSH) and activities of antioxidant enzymes in the kidney homogenates. Nitric oxide (NO) levels of urine and renal tissue were significantly inhibited in silymarin pre-treated animals. Furthermore, silymarin administration significantly inhibited the reduction of kidney content of adenosine triphosphate (ATP) parcels associated with this nephropathy. These results suggest that the protective role of silymarin in the prevention of APAP-induced nephrotoxicity in rats was associated with the decrease of oxidative and nitrosative stress in renal tissue as well as its capacity to improve the mitochondrial energy production. Thus, one could conclude that silymarin might be considered for the treatment of APAP-induced nephrotoxicity in rats. However, clinical studies are warranted to investigate such an effect in human subjects.