Physalin B (PB) is one of the major constituents of Physalis alkekengi var. franchetii, a well-known Chinese traditional herb. In this study, we demonstrated for the first time that PB exhibits significant antiproliferative and apoptotic activity in A549 human lung cancer cells in a concentration-dependent and time-dependent manner. Flow cytometric analyses indicated that PB-induced G2/M arrest through down-regulation of cyclin B1 and cell division control protein cyclin-dependent kinase 1, and up-regulation of p21. The reduction in the level of cyclin B1/cyclin-dependent kinase 1 complex down-regulated oxidative phosphorylation multisubunit activity to reduce mitochondrial energetic homeostasis. Moreover, defects in mitochondrial ATP synthesis and mitochondrial membrane potential were found in PB-treated cell lines. These abnormalities led to an increase in intracellular superoxide and apoptosis. Thus, as an inhibitor of mitochondrial energetic homeostasis, PB demonstrates potent antitumor activities and may be developed as an alternative therapeutic agent against non-small-cell lung cancer.