Reactive oxygen species (ROS) play an important role in cellular function via the activation of signaling cascades. ROS have been shown to affect mitochondrial biogenesis, morphology, and function. Their beneficial effects are likely mediated via the upregulation of transcriptional regulators such as peroxisome proliferator-activated receptor-γ coactivator-1 protein-α (PGC-1α). However, the ROS signals that regulate PGC-1α transcription in skeletal muscle are not understood. Here we examined the effect of H2O2 on the regulation of PGC-1α expression, and its relationship to AMPK activation. We demonstrate that 24 h of exogenous H2O2 treatment increased PGC-1α promoter activity and mRNA expression. Both effects were blocked with the addition of N-acetylcysteine, a ROS scavenger. These effects were mediated, in part, via upstream stimulatory factor-1/Ebox DNA binding and involved 1) interactions with downstream sequences and 2) the activation of AMPK. Elevated ROS led to the activation of AMPK, likely via a decline in ATP levels. The activation of AMPK using 5-aminoimidazole-4-carboxamide-1-β- d-ribofuranoside increased PGC-1α promoter activity and mRNA levels but reduced ROS production. Thus the net effect of AMPK activation on PGC-1α expression was a result of increased transcriptional activation, counterbalanced by reduced ROS production. The effects of H2O2 on PGC-1α expression differed depending on the level of ROS within the cell. Low levels of ROS result in reduced PGC-1α mRNA in the absence of an effect on PGC-1α promoter activation. In contrast, elevated levels of H2O2 induce PGC-1α transcription indirectly, via AMPK activation. These data identify unique interactions between ROS and AMPK activation on the expression of PGC-1α in muscle cells.