The growth of cancer cells is limited by energy supply which is regulated by the energy-sensor AMP-kinase (AMPK). In the present study the impact of AMPK activation on cell growth and apoptosis of DU-145 prostate cancer cells was investigated. Incubation with the AMPK activator aminoimidazole carboxamide ribonucleotide (AICAR) dose-dependent inhibited cell growth, activated AMPK, and inhibited mTOR. Furthermore AICAR treatment activated c-Jun-N-terminal kinase (JNK) and caspase-3, thereby initiating apoptosis. Within 60 min of treatment AICAR raised intracellular reactive oxygen species (ROS) which could be abolished in the presence of the free radical scavenger N-(2-mercaptopropionyl)glycin (NMPG), the AMPK inhibitor compound C and the respiratory chain complex I inhibitor rotenone, but not by the NADPH oxidase inhibitor VAS2870. Inhibition of ROS generation abolished AMPK activation by AICAR as well as JNK and caspase-3 activation. In summary our data demonstrate that activation of AMPK by AICAR induces apoptosis of prostate cancer cells by a signaling pathway involving ROS and activation of JNK.