Various isoforms of adiponectin circulate in the plasma. We purified high molecular weight (HMW) adiponectin from human plasma. HMW adiponectin was observed to activate AMP-activated protein kinase (AMPK), thereby increasing the phosphorylation of eNOS and NO production in endothelial cells. On the other hand, cells preincubated with HMW adiponectin had reduced TNFalpha-induced NF-kappaB activation. HMW adiponectin by itself was found to modestly activate NF-kappaB, which was significantly enhanced by inhibition of AMPK/eNOS activation. Thus, HMW adiponectin might have dual action, both pro and anti-inflammatory. An initial period of NF-kappaB activation by HMW adiponectin might be proinflammatory, but it could be counteracted by activation of AMPK/eNOS, which lead to a potential reduction in a second activation of NF-kappaB against inflammatory stimuli.