Recent in vitro studies suggest that adenosine monophosphate (AMP)‐activated protein kinase (AMPK) exerts inhibitory effects on cardiac hypertrophy. However, it is unclear whether long‐term activation of AMPK will affect cardiac hypertrophy in vivo. In these reports, we investigate the in vivo effects of long‐term AMPK activation on cardiac hypertrophy and the related molecular mechanisms. To examine the effects of AMPK activation in the development of pressure overload‐induced cardiac hypertrophy, we administered 5‐aminoimidazole 1 carboxamide ribonucleoside (AICAR, 0.5 mg/g body wt), a specific activator of AMPK, to rats with transaortic constriction (TAC) for 7 weeks. We found that long‐term AMPK activation attenuated cardiac hypertrophy, and improved cardiac function in rats subjected to TAC. Furthermore, long‐term AMPK activation attenuated protein synthesis, diminished calcineurin‐nuclear factor of activated T cells (NFAT) and nuclear factor κB (NF‐κB) signaling in pressure overload‐induced hypertrophic hearts. Our in vitro experiments further proved that activation of AMPK by infection of AdAMPK blocked cardiac hypertrophy and NFAT, NF‐κB, and MAPK signal pathways. The present study demonstrates for the first time that pharmacological activation of AMPK inhibits cardiac hypertrophy in through blocking signaling transduction pathways that are involved in cardiac growth. It presents a potential therapy strategy to inhibit pathological cardiac hypertrophy by increasing the activity of AMPK. J. Cell. Biochem. 100: 1086–1099, 2007. © 2007 Wiley‐Liss, Inc.