Rhus verniciflua STOKES (RVS) is used as an anti-cancer agent in traditional herbal medicine. However, the underlying molecular mechanism of its action is poorly understood. Here, we elucidated the mechanism of the anti-cancer mechanism of RVS in MCF-7 human breast cancer cells. We found that RVS increased the phosphorylation of AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC) and suppressed cell viability in an AMPK-dependent fashion. RVS also induced an increase in reactive oxygen species (ROS) levels. RVS-induced AMPK phosphorylation was not observed in the presence of N-acetyl-cysteine (NAC), which indicated that ROS is associated with RVS-induced AMPK phosphorylation. In addition, fluorescent staining (Annexin V/propidium iodide) revealed that RVS increased the expression of Annexin V, which indicates that RVS leads to cancer-induced apoptosis. Moreover, RVS increased the phosphorylation of p53 and the expression of Bax. The inhibition of AMPK blocked RVS-induced p53 phosphorylation and Bax expression, which suggests that AMPK is involved in RVS-induced cancer apoptosis. Taken together, these results demonstrate that RVS has anti-tumor effects on MCF-7 cells through an AMPK-signaling pathway.