Peroxisomes are indispensable organelles for lipid metabolism in humans and their biogenesis has been assumed to be under regulation by peroxisome proliferator-activated receptors (PPARs). However, recent studies in hepatocytes suggest that the mitochondrial proliferator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) also acts as an upstream transcriptional regulator for enhancing peroxisomal abundance and associated activity. It is unknown whether the regulatory mechanism(s) for enhancing peroxisomal function is through the same node as mitochondrial biogenesis in human skeletal muscle (HSkM) and whether fatty acid oxidation (FAO) is affected. Primary myotubes from vastus lateralis biopsies from lean donors (BMI =24.0 ± 0.6 kg/m², N = 6) were exposed to adenovirus encoding human PGC-1α or GFP control. Peroxisomal biogenesis proteins (Peroxins) and genes (PEXs) responsible for proliferation and functions were assessed by western blotting and real-time qRT-PCR respectively. 1-¹⁴C palmitic acid and 1-¹⁴C lignoceric acid (exclusive peroxisomal specific substrate) were used to assess mitochondrial oxidation of peroxisomal derived metabolites. Following overexpression of PGC-1α, 1) Peroxisomal membrane protein 70kD (PMP70), PEX19, and mitochondrial citrate synthetase protein content were significantly elevated (P<0.05) 2) PGC-1α, PMP70, key PEXs, and peroxisomal β-oxidation mRNA expression levels were significantly upregulated (P<0.05) and 3) A concomitant increase in lignoceric acid oxidation by both peroxisomal and mitochondrial activity was observed (P<0.05). These novel findings demonstrate that, in addition to the proliferative effect on mitochondria, PGC-1α can induce peroxisomes and accompanying elevations in long-chain and very-long-chain fatty acid oxidation by a peroxisomal-mitochondrial functional cooperation as observed in HSkM cells.