Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classical glycolytic enzyme that is involved in cellular energy production and has important housekeeping functions. We used the natural prooxidant and proatherogenic molecule oxidized low-density lipoprotein (OxLDL) to determine a potential link between OxLDL-promoted oxidative stress, GAPDH expression, and smooth muscle cell energy metabolism. OxLDL but not native LDL (nLDL) produced a 60% to 100% dose- and time-dependent reduction of GAPDH protein. OxLDL increased reactive oxygen species (ROS) formation, including rapid elevation of H(2)O(2) levels. OxLDL decreased intracellular catalase expression, likely contributing to the increase in H(2)O(2). Antioxidants, anti-CD36 receptor antibody, NADPH oxidase, or lipoxygenase blockers decreased OxLDL-specific ROS and prevented GAPDH downregulation. 12/15-Lipoxygenase or p47phox deficiency resulted in attenuation of GAPDH downregulation, but 5-lipoxygenase suppression had no effect. OxLDL or exogenous H(2)O(2) oxidized GAPDH thiols, decreasing GAPDH protein half-life and increasing GAPDH sensitivity to proteasome-mediated protein degradation in vitro. OxLDL- or small interfering RNA-specific downregulation of GAPDH resulted in 65% reduction in glycolysis rate and 82% decrease in ATP levels. In conclusion, our data demonstrate that OxLDL downregulated GAPDH via a H(2)O(2)-dependent decrease in protein stability. GAPDH protein damage resulted in marked depletion of cellular ATP levels. Our data have important implications for understanding the metabolic effect of OxLDL on the vessel wall and mechanism of atherogenesis.