UCP3 (uncoupling protein-3) is a mitochondrial membrane transporter expressed preferentially in skeletal muscle. UCP3 lowers mitochondrial membrane potential and protects muscle cells against an overload of fatty acids, and it probably reduces excessive production of reactive oxygen species. Accordingly, ucp3 gene transcription is highly sensitive to fatty acid-dependent stimulation and also to other unrelated stress signals. In this study, glucocorticoids are identified as major inducers of ucp3 gene transcription in muscle. Glucocorticoids activate the transcription of the ucp3 gene through a glucocorticoid receptor-binding site in the promoter region. Glucocorticoids are capable of inducing ucp3 gene transcription independently from the myogenic regulatory factor MyoD, in contrast with the transcriptional activation of the ucp3 gene through other nuclear hormone receptors. An interplay of regulatory factors modulates positively (p300) or negatively (histone deacetylases) the action of glucocorticoids on ucp3 gene transcription via histone acetylation or deacetylation processes, respectively. Among them, SIRT1 acts as a major repressor of ucp3 gene expression in response to glucocorticoids. The action of SIRT1 requires its deacetylase activity and results in histone deacetylation in the ucp3 promoter. Moreover, it involves a specific impairment of association of p300 with the glucocorticoid receptor. Agents activating SIRT1, such as resveratrol, repress ucp3 gene expression. The control of SIRT1 activity via the metabolic redox status of the cell points to a novel regulatory pathway of ucp3 gene transcription in response to metabolic and stress signaling in muscle cells.