Nitric oxide (NO) has both prooxidant and antioxidant activities in the endothelium; however, the molecular mechanisms involved are still a matter of controversy. PGC‐1α [peroxisome proliferators‐activated receptor (PPAR) γ coactivator 1‐α] induces the expression of several members of the mitochondrial reactive oxygen species (ROS) detoxification system. Here, we show that NO regulates this system through the modulation of PGC‐1α expression. Short‐term (<12 h) treatment of endothelial cells with NO donors down‐regulates PGC‐1α expression, whereas long‐term (>24 h) treatment up‐regulates it. Treatment with the NOS inhibitor L‐NAME has the opposite effect. Down‐regulation of PGC‐1α by NO is mediated by protein kinase G (PKG). It is blocked by the soluble guanylate cyclase (sGC) inhibitor ODQ and the PKG inhibitor KT5823, and mimicked by the cGMP analog 8‐Br‐cGMP. Changes in PGC‐1α expression are in all cases paralleled by corresponding variations in the mitochondrial ROS detoxification system. Cells that transiently overexpress PGC‐1α from the cytomeglovirus (CMV) promoter respond poorly to NO donors. Analysis of tissues from eNOS^−/− mice showed reduced levels of PGC‐1α and the mitochondrial ROS detoxification system. These data suggest that NO can regulate the mitochondrial ROS detoxification system both positively and negatively through PGC‐1α. —Borniquel, S., Valle, I., Cadenas, S., Lamas, S., and Monsalve, M. Nitric oxide regulates mitochondrial oxidative stress protection via the transcriptional coactivator PGC‐1α. FASEB J. 20, E1216–E1227 (2006)