OBJECTIVE: Increased DNA methylation of the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) has been reported in skeletal muscle from type 2 diabetes (T2D) subjects and from low birth weight (LBW) subjects with an increased risk of T2D. High-fat overfeeding increases PPARGC1A DNA methylation in muscle in a birth weight dependent manner. However, PPARGC1A DNA methylation in subcutaneous adipose tissue (SAT) in LBW subjects has not previously been investigated. Our objective was to determine PPARGC1A DNA methylation and mRNA expression in basal and insulin-stimulated SAT from LBW and matched normal birth weight (NBW) subjects during control and high-fat overfeeding. MATERIALS/METHODS: Nineteen young healthy men with LBW and 26 NBW controls were studied after both a 5-day high-fat overfeeding and a control diet in a randomized crossover setting. DNA methylation was assessed with bisulfite sequencing and mRNA expression with quantitative real-time PCR. RESULTS: Following high-fat overfeeding, increased SAT PPARGC1A DNA methylation was observed in LBW subjects but not in NBW controls. Basal SAT PPARGC1A mRNA expression was unaffected by diet and similar in the two groups. However, LBW subjects showed an increased SAT PPARGC1A mRNA expression during insulin-stimulation. SAT PPARGC1A methylation correlated inversely with mRNA expression during insulin-stimulation. CONCLUSIONS: The study adds to the increasing awareness of PPARGC1A DNA methylation being flexible and influenced by high-fat overfeeding in a birth weight dependent manner with muscle and fat responding differently. Further data are needed to understand the role of PPARGC1A DNA methylation in insulin resistance and developmental programming of T2D.