Nitric oxide (NO) is a small molecule with distinct roles in diverse physiological functions in biological systems, among them the control of the apoptotic signalling cascade. By combining proteomic, genetic and biochemical approaches we demonstrate that NO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are crucial mediators of yeast apoptosis. Using indirect methodologies and a NO-selective electrode, we present results showing that H2O2-induced apoptotic cells synthesize NO that is associated to a nitric oxide synthase (NOS)-like activity as demonstrated by the use of a classical NOS kit assay. Additionally, our results show that yeast GAPDH is a target of extensive proteolysis upon H2O2-induced apoptosis and undergoes S-nitrosation. Blockage of NO synthesis with Nomega-nitro-L-arginine methyl ester leads to a decrease of GAPDH S-nitrosation and of intracellular reactive oxygen species (ROS) accumulation, increasing survival. These results indicate that NO signalling and GAPDH S-nitrosation are linked with H2O2-induced apoptotic cell death. Evidence is presented showing that NO and GAPDH S-nitrosation also mediate cell death during chronological life span pointing to a physiological role of NO in yeast apoptosis.