Contrast-induced nephropathy accounts for >10% of all causes of hospital-acquired renal failure, causes a prolonged in-hospital stay and represents a powerful predictor of poor early and late outcome. Mechanisms of contrast-induced nephropathy are not completely understood. In vitro data suggests that contrast media (CM) induces a direct toxic effect on renal tubular cells through the activation of the intrinsic apoptotic pathway. It is unclear whether this effect has a role in the clinical setting. In this work, we evaluated the effects of CM both in vivo and in vitro. By analyzing urine samples obtained from patients who experienced contrast-induced acute kidney injury (CI-AKI), we verified, by western blot and immunohistochemistry, that CM induces tubular renal cells apoptosis. Furthermore, in cultured cells, CM caused a dose-response increase in reactive oxygen species (ROS) production, which triggered Jun N-terminal kinases (JNK1/2) and p38 stress kinases marked activation and thus apoptosis. Inhibition of JNK1/2 and p38 by different approaches (i.e. pharmacological antagonists and transfection of kinase-death mutants of the upstream p38 and JNK kinases) prevented CM-induced apoptosis. Interestingly, N-acetylcysteine inhibited ROS production, and thus stress kinases and apoptosis activation. Therefore, we conclude that CM-induced tubular renal cells apoptosis represents a key mechanism of CI-AKI.