Metformin is known to increase lipoprotein lipase (LPL) mass level in serum. Lipoprotein lipase is produced by adipose tissue and skeletal muscles. This study aimed to examine the effect of metformin on LPL production in adipocytes and skeletal muscle cells and to investigate the mechanism by which metformin enhances LPL production. 3T3-L1 preadipocytes and L6 skeletal muscle cells were incubated with metformin or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Lipoprotein lipase activity, LPL protein expression, and LPL messenger RNA (mRNA) expression were measured. Metformin increased LPL activity only in skeletal muscle cells. To clarify the mechanism of this phenomenon, AICAR, which is well known as an activator of adenosine monophosphate-activated protein kinase (AMPK), was used. Metformin and AICAR enhanced phosphorylated AMPK in skeletal muscle cells by Western blot analysis. Like metformin, AICAR increased LPL activity only in skeletal muscle cells. Both metformin and AICAR also enhanced LPL protein and LPL mRNA expressions in skeletal muscle cells but not in adipocytes. Phosphorylated AMPK protein expression was decreased when AMPK signaling was interfered by AMPKalpha small interfering RNA. Lipoprotein lipase activity and LPL expression, which were enhanced by 1 mumol/L metformin, were reduced by AMPKalpha small interfering RNA. These results suggest that metformin increases LPL activity, LPL protein expression, and LPL mRNA expression through activation of AMPK in skeletal muscle cells but not in adipocytes.