Hypoxia induces oxidative damage in skeletal muscle. Uncoupling protein 3 (UCP3) is the skeletal muscle enriched uncoupling protein and has previously been shown to confer resistance against oxidative stress. We show that hypoxia robustly upregulates skeletal muscle UCP3 and that the absence of UCP3 in primary skeletal myocytes exacerbates hypoxia induced reactive oxygen species generation. In this context, we reasoned that the investigation of the regulation of UCP3 may identify novel hypoxia-responsive regulatory pathways that modulate intrinsic anti-oxidant defenses. By screening a transcription factor array of 704 full-length cDNAs in murine C2C12 myoblasts following cotransfection of a murine UCP3 promoter-luciferase construct and myoD we identified numerous candidate regulatory factors that upregulate UCP3. Active transcription factor-1 (ATF-1) was identified and as this transcription factor is a known component of a multiprotein hypoxia-induced regulatory complex, we explored its role in hypoxia-mediated UCP3 upregulation. Site-directed mutagenesis and chromatin immunoprecipitation assays identify a 10 bp region required for ATF-1 induction of UCP3 promoter activity. Hypoxia promotes the phosphorylation of ATF-1 and the knockdown of ATF-1 by shRNA prevents hypoxia-mediated upregulation of UCP3. Pharmacologic inhibition of p38MAP kinase prevents both hypoxia-mediated ATF-1 phosphorylation and UCP3 upregulation. PKA signaling does not modulate hypoxia-induced UCP3 upregulation and neither does HIF-1alpha activation by cobalt chloride. In conclusion ATF-1, via p38MAP kinase activation, functions as a novel regulatory pathway driving UCP3 expression. These data reinforce the role of ATF-1 as a hypoxia-responsive trans-activator and identifies a novel regulatory program that may modulate cellular responses to oxygen-deficit.