Silibinin is an active constituent extracted from the blessed milk thistle (Silybum marianum). In a previous study, we demonstrated that silibinin treatment induced the generation of reactive nitrogen species (RNS), which were associated with reactive oxygen species (ROS), and caused apoptosis and autophagy in HeLa cells. Another study reported that silibinin treatment attenuated the apoptotic effect of sodium nitroprusside (SNP) by generating ROS in rat pheochromocytoma PC12 cells [ 1 ]. To clarify the relationship between RNS and nitric oxide (NO) in HeLa cells, we chose SNP as a NO donor to inhibit the cell viability. We found that silibinin treatment did not reduce the cytotoxicity of NO by reducing the ROS-induced RNS levels; conversely, silibinin treatment enhanced the cytotoxicity of NO. Pre-treatment with the NO scavenger PTIO preserved the viability of SNP- or silibinin-treated cells. Buthionine sulfoximine (BSO) treatment was also used to deplete the level of glutathione (GSH) and subsequently enhance the cytotoxicity of NO. Pre-treatment with BSO enhanced the SNP-induced reduction of cell viability but had no such effects in the silibinin-treated cells. These results led us to investigate whether silibinin treatment could induce the depletion of GSH. JNK and p53 have been shown to mediate the depletion of GSH [ 2 , 3 ], and we previously demonstrated the existence of a ROS-JNK-p53 cycle in silibinin-treated HeLa cells [ 4 ]. Thus, we speculated that p53 also plays a crucial role in the silibinin-induced GSH depletion. To elucidate the role of p53 in this process, A431 cells were used because they are naturally devoid of a functional p53 (p53His273 mutation). To our surprise, silibinin treatment did not lower the GSH level in A431 cells but rather elevated the GSH level. Unlike the ROS level, the NO level was still up-regulated by silibinin treatment in A431 cells. Cumulatively, these findings support the idea that the silibinin-induced GSH depletion, which is mediated by p53, enhances the cytotoxicity of NO in HeLa cells.