Sodium nitroprusside (SNP) is a water-soluble iron nitrosyl complex clinically used as a powerful vasodilator for treatment of hypertension; and, in basic research, it has been used to mainly investigate the cytotoxic effects of nitrosative stress. Although NO is considered a pharmacologically active molecule, not all of the biological effects of SNP are dependent on its NO moiety. To elucidate the molecular executioner(s) responsible for SNP cytotoxicity, this study determines the involvement of oxidative stress in p53 activation and apoptotic induction elicited by SNP in SH-SY5Y neuroblastoma cells. We demonstrate that proapoptotic activity of SNP is independent of NO production, because SNP and its 2-day light-exhausted compound SNP(ex) trigger apoptosis to the same extent. We provide evidence for the occurrence of oxidative stress and oxidative damage during both SNP and SNP(ex) exposure and demonstrate that iron-derived reactive oxygen species (ROS) are the genuine mediators of their cytotoxicity. We show that p53 is equally activated upon both SNP and SNP(ex) treatments. Moreover, as demonstrated by small interfering RNA experiments, we indicate its primary role in the induction of apoptosis, suggesting the ineffectiveness of NO in its engagement. The attenuation of p53 levels, obtained by oxy-radical scavengers, is consistent with the recovery of cell viability and ROS decrease, demonstrate that SNP-mediated p53 activation is an event triggered by ROS and/or ROS-mediated damages. Together, our results suggest that investigations of the physiopathological effects of SNP should consider the role of ROS, other than NO, particularly in some conditions such as apoptotic induction and p53 activation.