Agaricus blazei is well known as a traditional medicinal mushroom and it has been shown to exhibit immunostimulatory and anti-cancer activity. However, the cellular and molecular mechanism of apoptosis of cancer cells is poorly understood. In this study, we have investigated whether A. blazei extract (ABE) exerts anti-proliferative and apoptotic effects on human leukemia THP-1 cells. It was found that ABE induced a time- and dose-dependent increase in leukemia cells apoptosis through caspase-3 activation and PARP cleavage. Activation of caspase- 9 induced by ABE suggested that ABE-induced signaling was mediated through a mitochondrial death pathway. In addition, we observed an elevation of ROS and a consequent loss of mitochondrial membrane potential, further suggesting that ABE-induced death signaling was mediated through a mitochondrial oxygen stress pathway. The antioxidant Nacetylcysteine, however, opposed ABE-mediated mitochondrial dysfunction, caspase activation, and apoptosis, supporting the role of ROS in the apoptotic process. We conclude that ABE induces apoptosisin human leukemia cells through a reactive oxygen species and caspase-dependent mitochondrial pathway.