We investigated whether AMP‐activated protein kinase (AMPK), a multi‐functional regulator of energy homeostasis, participates in the regulation of erythropoietin (EPO)‐mediated activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) and mice. In ECs, treatment with EPO increased the phosphorylation of AMPK, acetyl‐CoA carboxylase (ACC), and eNOS, as revealed by Western blot analysis. Inhibition of AMPK activation by compound C or dominant‐negative AMPK mutant abrogated the EPO‐induced increase in the phosphorylation of AMPK, ACC, and eNOS, as well as nitric oxide (NO) production. Additionally, suppression of AMPK activation abolished EPO‐induced EC proliferation, migration and tube formation. Immunoprecipitation analysis demonstrated that AMPK mediated the EPO‐induced increase in the phosphorylation of β common receptor (βCR) and the formation of a βCR–AMPK–eNOS complex. In mice, inhibition of AMPK activation by compound C markedly decreased EPO‐elicited angiogenesis in Matrigel plugs. Furthermore, the phosphorylation of AMPK and eNOS was significantly higher in aortas from EPO transgenic mice than wild‐type mice. Moreover, treatment with EPO neutralizing antibody greatly reduced the exercise training‐induced increase in phosphorylation of AMPK and eNOS in aortas of wild‐type mice. Taken together, EPO may trigger AMPK‐dependent signaling, which leads to enhanced phosphorylation of βCR and eNOS, increased βCR–AMPK–eNOS complex formation, NO production, and, ultimately, angiogenesis. J. Cell. Physiol. 227: 3053–3062, 2012. © 2011 Wiley Periodicals, Inc.