Although the classical redox functions of co-enzyme NAD(+) are firmly established in metabolism, there are numerous enzymes that catalyze cleavage of NAD(+) to yield free ADP-ribose (ADPr) or related metabolites, whose functions remain largely unknown. Here we show that the Nudix (nucleoside diphosphate linked to another moiety X) hydrolase Ysa1 from Saccharomyces cerevisiae is a major regulator of cellular ADPr and O-acetyl-ADP-ribose (OAADPr). OAADPr is the direct product of NAD(+)-dependent protein deacetylases (sirtuins) and is readily converted to ADPr. Ysa1 cleaves ADPr/OAADPr into ribose phosphate/acetyl-ribose phosphate and AMP. In cells lacking Ysa1 (Deltaysa1), ADPr and OAADPr levels increased approximately 50%, with a corresponding decrease in AMP. Strikingly, Deltaysa1 cells display higher resistance to exogenous reactive oxygen species (ROS) and 40% lower basal levels of endogenous ROS, compared with wild type. The biochemical basis for these differences in ROS-related phenotypes was investigated, and the results provide evidence that increased ADPr/OAADPr levels protect cells via the following two pathways: (i) lower ROS production through inhibition of complex I of the mitochondrial electron transport chain, and (ii) generation of higher levels of NADPH to suppress ROS damage. The latter occurs through diverting glucose into the pentose phosphate pathway by ADPr inhibition of glyceraldehyde-3-phosphate dehydrogenase, a central enzyme of glycolysis.