In Taiwan, oral cancer is the fourth leading cause of male cancer mortality, and is still increasing. The Basiodiomycete, Agaricus brasiliensis Murill (ABM) is a dietary mushroom and has been known for its immuneenhancing, antitumor, antioxidation, antiviral and antimutagenesis functions. However, the exact anticancer mechanisms of ABM on human oral cancer cells are still unclear. In the present study, we investigated the effects of 50% ethanol crude extracts and hot water extracts of ABM on oral cancer CAL 27 cells. We observed that 0.9 mg/ml and 0.7 mg/ml of ABM 50% ethanol crude extracts and hot water, respectively, caused morphological changes and significantly reduced cell viability after 48-h treatment. The results showed that both extracts of ABM inhibited cell proliferation, increased the Ca(2+) release, reduced the mitochondria membrane potential (ΔΨm), and caused cell cycle arrest in the G(0)/G(1) phase, which contributed to apoptosis. Additionally, ABM induced DNA fragmentation, a characteristic of apoptosis and the expressions of apoptosis-related proteins, including apoptosis-inducing factor, cytochrome c, and caspase-3, were increased. Overall, we demonstrated that 50% ethanol crude extract and hot water extracts of ABM were able to induce apoptotic cell death in CAL 27 cells via the release of cytochrome c from mitochondria into the cytoplasm and activation of caspase-3 in vitro.