|Adiponectin is a 30-kD protein secreted by adipose tissue under normal conditions. Serum adiponectin concentrations are decreased in obesity and type 2 diabetes. AdipoR1 and AdipoR2 are two receptors of adiponectin with AdipoR1 abundantly expressed in muscle and AdipoR2 predominantly expressed in liver. Adiponectin has been demonstrated to decrease hepatic glucose production by inhibiting hepatic G-6-Pase and PEPCK mRNA expression, but its effects on hepatic glycogen synthesis have not been examined. In addition, eukaryotically and bacterially expressed adiponectin may have different effects on glucose metabolism due to some important structural differences. In this study, we investigated the effects of eukaryotically and bacterially expressed adiponectin on hepatic glycogen synthesis as well as the mechanisms involved in this process. We also compared the effects of these two sources of adiponectin on muscle basal glucose uptake. A human hepatoma cell line (HepG2) and a rat ventricular cell line (H9c2) were utilized in our experiments. Hepatic basal and insulin-stimulated glycogen synthesis was evaluated in HepG2 cells treated for 3 h with eukaryotically expressed adiponectin (1 µg/ml and 5µg/ml), bacterially expressed adiponectin (20 µg/ml and 30 µg/ml), 1nM insulin and 1 nM insulin plus eukaryotically expressed adiponectin (1 µg/ml and 5µg/ml). Also, HepG2 cells were used to determine if adiponectin impacted insulin receptor signaling. Muscle glucose uptake was evaluated in H9c2 cells treated for 10 min with the same concentrations of eukaryotically and bacterially expressed adiponectin. The results indicated that AdipoR2 is present in HepG2 cells by western blotting and real-time PCR, and its expression is inhibited by insulin. AdipoR1 mRNA is also verified to be present in H9c2 cells by real-time PCR. For the metabolic effects, eukaryotically but not bacterially expressed adiponectin inhibits basal and insulin-stimulated glycogen synthesis in HepG2 cells, while bacterially but not eukaryotically expressed adiponectin stimulates basal glucose uptake in H9c2 cells. Importantly, adiponectin does not utilize the insulin signal pathway to produce the effect on hepatic glycogen synthesis, as IRS-1 was not phosphorylated by adiponectin in HepG2 cells. Our data suggest that multimer formation of higher order structure is very important for adiponectin’s function in liver but not in muscle. A major effect of adiponectin on hepatic glucose metabolism may be to inhibit glycogen synthesis thereby providing more glucose for glycolysis and ATP generation.