Cysteine S-nitrosylation is a posttranslational modification by which nitric oxide regulates protein function and signaling. Studies of individual proteins have elucidated specific functional roles for S-nitrosylation, but knowledge of the extent of endogenous S-nitrosylation, the sites that are nitrosylated, and the regulatory consequences of S-nitrosylation remains limited. We used mass spectrometry–based methodologies to identify 1011 S-nitrosocysteine residues in 647 proteins in various mouse tissues. We uncovered selective S-nitrosylation of enzymes participating in glycolysis, gluconeogenesis, tricarboxylic acid cycle, and oxidative phosphorylation, indicating that this posttranslational modification may regulate metabolism and mitochondrial bioenergetics. S-nitrosylation of the liver enzyme VLCAD [very long chain acyl–coenzyme A (CoA) dehydrogenase] at Cys²³⁸, which was absent in mice lacking endothelial nitric oxide synthase, improved its catalytic efficiency. These data implicate protein S-nitrosylation in the regulation of β-oxidation of fatty acids in mitochondria.