Physalin B (PB), one of the major active steroidal constituents of Cape gooseberry (Physalis alkekengi L.), possesses a wide spectrum of biological activities. Although the anticancer activity of PB was reported in previous studies, the underlying mechanisms are still not well stated. In this study, the anticancer effect and the underlying mechanisms of PB were investigated in breast cancer cells. PB significantly reduced the viability of three human breast cancer cell lines, MCF-7, MDA-MB-231 and T-47D, in a concentration-and time-dependent manner. PB induced cell cycle arrest at G2/M phase and promoted cleavage of PARP (poly (ADP-ribose) polymerase), caspases 3, caspase 7 and caspase 9 to stimulate cell apoptosis. Further studies showed that PB induced breast cancer cells apoptosis in a p53-dependent manner in MCF-7 cells. PB also suppressed the phosphorylation of Akt (protein kinase B) and PI3K (phosphoinositide 3-kinase), and increased the phosphorylation of GSK-3 beta (glycogen synthase kinase 3 beta). Taken together, our results indicated that PB might serve as a potential therapeutic agent for breast cancer.