The present study determined whether AMP-activated protein kinase (AMPK) regulates heme oxygenase (HO)-1 gene expression in endothelial cells (ECs) and if HO-1 contributes to the biological actions of this kinase. Treatment of human ECs with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) stimulated a concentration- and time-dependent increase in HO-1 protein and mRNA expression that was associated with a prominent increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) protein. Induction of HO-1 was also observed in rat carotid arteries after the in vivo application of AICAR. Induction of HO-1 by AICAR was blocked by the AMPK inhibitor compound C, the adenosine kinase inhibitor 5'-iodotubercidin, and by silencing AMPK-α(1/2) and was mimicked by the AMPK activator A-769662 and by infecting ECs with an adenovirus expressing constitutively active AMPK-α(1). AICAR also induced a significant rise in HO-1 promoter activity that was abolished by mutating the antioxidant responsive elements of the HO-1 promoter or by the overexpression of dominant negative Nrf2. Finally, activation of AMPK inhibited cytokine-mediated EC death, and this was prevented by the HO inhibitor tin protoporphyrin-IX or by silencing HO-1 expression. In conclusion, AMPK stimulates HO-1 gene expression in human ECs via the Nrf2/antioxidant responsive element signaling pathway. The induction of HO-1 mediates the antiapoptotic effect of AMPK, and this may provide an important adaptive response to preserve EC viability during periods of metabolic stress.