Doxorubicin is one of the most widely used anti-cancer drugs, but its clinical application is compromised by severe adverse effects in different organs including cardiotoxicity. In the present study we explored mechanisms of doxorubicin-induced cytotoxicity by revealing a novel role for the AMP-activated protein kinase alpha2 (AMPKalpha2) in mouse embryonic fibroblasts (MEFs). Doxorubicin robustly induced the expression of AMPKalpha2 in MEFs but slightly reduced AMPKalpha1 expression. Our data support the previous notion that AMPKalpha1 harbors survival properties under doxorubicin treatment. In contrast, analyses of Ampkalpha2(-/-) MEFs, gene knockdown of AMPKalpha2 by shRNA, and inhibition of AMPKalpha2 activity with an AMPK inhibitor indicated that AMPKalpha2 functions as a pro-apoptotic molecule under doxorubicin treatment. Doxorubicin induced AMPKalpha2 at the transcription level via E2F1, a transcription factor that regulates apoptosis in response to DNA damage. E2F1 directly transactivated the Ampkalpha2 gene promoter. In turn, AMPKalpha2 significantly contributed to stabilization and activation of E2F1 by doxorubicin, forming a positive signal amplification loop. AMPKalpha2 directly interacted with and phosphorylated E2F1. This signal loop was also detected in H9c2, C2C12, and ECV (human epithelial cells) cells as well as mouse liver under doxorubicin treatment. Resveratrol, which has been suggested to attenuate doxorubicin-induced cytotoxicity, significantly blocked induction of AMPKalpha2 and E2F1 by doxorubicin, leading to protection of these cells. This signal loop appears to be non-carcinoma-specific because AMPKalpha2 was not induced by doxorubicin in five different tested cancer cell lines. These results suggest that AMPKalpha2 may serve as a novel target for alleviating the cytotoxicity of doxorubicin.