Adiponectin (APN) exerts its metabolic regulation largely through AMP-dependent protein kinase (AMPK). However, the role of AMPK in adiponectin's anti-apoptotic effect in ischemic/reperfused (I/R) adult cardiomyocytes remains incompletely understood. The current study was designed to determine the involvement of AMPK in the anti-apoptotic signaling of adiponectin. Cardiomyocytes from adult male mice overexpressing a dominant negative ₂ subunit of AMPK (AMPK-DN) or wild-type (WT) littermates were subjected to simulated I/R (SI/R) and pretreated with 2 µg/ml gAPN or vehicle. SI/R-induced cardiomyocyte apoptosis was modestly increased in AMPK-DN cardiomyocytes (P<0.05). Treatment with gAPN significantly reduced SI/R-induced apoptosis in WT cardiomyocytes as well as in AMPK-DN cardiomyocytes, indicating that the anti-apoptotic effect of gAPN is partially AMPK-independent. Furthermore, gAPN-induced endothelial nitric oxide synthase (eNOS) phosphorylation was significantly reduced in AMPK-DN cardiomyocytes, suggesting the APN-eNOS signaling axis is impaired in AMPK-DN cardiomyocytes. Additional experiments demonstrated that treatment of AMPK-DN cardiomyocytes with gAPN reduced SI/R-induced NADPH oxidase overexpression, decreased superoxide generation, and blocked peroxynitrite formation to the same extent as that observed in WT cardiomyocytes. Collectively, our current study demonstrated that although the metabolic and eNOS activation effects of adiponectin is largely mediated by AMPK, the superoxide-suppressing effect of adiponectin is not mediated by AMPK, and this AMPK-independent anti-oxidant property of adiponectin increased nitric oxide bioavailability, and exerted significant anti-apoptotic effect.