Endurance exercise induces mitochondrial biogenesis in skeletal muscle. It has been shown that lipin-1 acts as a transcriptional coactivator in liver, and stimulates gene expression of mitochondrial enzymes. We hypothesized that lipin-1 might be involved in exercise-induced mitochondrial biogenesis in skeletal muscle. The present investigation first demonstrated that lipin-1 mRNA in rat triceps muscle was increased by approximately 2-fold after an acute bout of endurance swimming exercise. Second, ectopic expression of lipin-1 in L6 myotube increased carnitine palmitoyltransferase-1 and δ-aminolevulinate synthase gene expression. Finally, lipin-1 mRNA expression in rat triceps muscle was significantly elevated at 6 h after subcutaneous injections of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or clenbuterol, which are 5′-AMP-activated protein kinase (AMPK) and β2-adrenergic receptor (β2-AR) activators, respectively. These results may suggest that enhanced expression of lipin-1 is involved in exercise-induced mitochondrial enzyme adaptations, possibly through AMPK- and β2-AR-related mechanisms.