Luteolin, a plant-derived flavonoid, is thought to inhibit tumor growth. However, the precise molecular mechanisms by which luteolin inhibits cancer cell growth remain unclear. In the present study, we evaluated the role of AMP-activated protein kinase (AMPK) in the inhibition of cancer cell growth by luteolin in HepG2 hepatocarcinoma cells. AMPK is a metabolic sensor and may prevent carcinogenesis via modulation of signaling networks. We found that luteolin strongly induced cell death in HepG2 cells and dramatically reduced the tumor volume in a tumor xenograft model; both effects were accompanied by AMPK activation by luteolin. Luteolin also had a strong inhibitory effect on nuclear factor (NF)-κB. To determine the relationship between AMPK and NF-κB signaling, we used Compound C, a pharmacological AMPK inhibitor, and a dominant-negative form of AMPK. Our results indicated that inhibition of AMPK activity restored luteolin-inhibited NF-κB DNA-binding activity. These results suggest that AMPK activity is critical for the inhibition of cancer cell growth, possibly via modulation of NF-κB activity. We also showed that luteolin treatment causes the release of reactive oxygen species (ROS) and that these intracellular ROS in turn mediate AMPK-NF-κB signaling in HepG2 hepatocarcinoma cells. In conclusion, we propose that AMPK is a novel regulator of NF-κB in luteolin-induced cancer cell death. Furthermore, our results suggest that AMPK is an attractive target for cancer prevention by flavonoids.