Extracellular signal-regulated kinase (ERK) belongs to the mitogen-activated protein kinases (MAPK) superfamily. Aberrant upregulation and activation of ERK cascades may often lead to tumor cell development. However, how ERK is involved in tumor progression is yet to be defined. In current study, we described that ERK undergoes S-nitrosylation by nitric oxide (NO). ERK S-nitrosylation inhibits its phosphorylation and triggers apoptotic program as verified by massive apoptosis in fluorescence staining. The proapoptotic effect of NO induced S-nitrosylation is reversed by NO scavenger Haemoglobin (HB). Furthermore, an S-nitrosylation dead ERK mutant C183A also demolishes the proapoptotic potential of NO and favors cell survival. Therefore, Cys(183) might be a potential S-nitrosylation site in ERK. In addition, S-nitrosylation is a general phenomenon that regulates ERK activity. These findings identify a novel link between NO-mediated S-nitrosylation and ERK regulation, which provide critical insights into the control of apoptosis and tumor development.