The anti-cancer effects and possible mechanisms of the freshwater clam (Corbicula fluminea Muller) and its active compounds (FME) on cell viability in human leukemia HL-60 cells were investigated. This study demonstrated that FME was able to inhibit cell proliferation in a concentration- and time-dependent manner. Treatment with FME caused induction of caspase-2, caspase-3, caspase-6, caspase-8, and caspase-9 activity in a time-dependent manner, but not affect caspase-1 activity; it induced the proteolysis of DNA fragmentation factor (DFF-45) and poly(ADP-ribose) polymerase (PARP). Induction of cell death by FME was completely prevented by a pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) and a caspase-2 inhibitor, Z-Val-Asp-Val-Ala-Asp-FMK (Z-VDVAD-FMK). Furthermore, treatment with FME caused a rapid loss of mitochondrial transmembrane potential, stimulation of generation of reactive oxygen species (ROS), release of mitochondrial cytochrome c into cytosol, and GSH depletion. Anti-oxidants such as N-acetylcysteine, catalase, superoxide dismutase, allopurinol, and pyrrolidine dithiocarbamate, but not diphenylene iodonium, significantly inhibited FME-induced cell death. In addition, the results showed that FME-induced apoptosis was accompanied by up-regulation of Bax and Bad, and down-regulation of Bcl-2 and Bcl-XL. Taken together, induction of apoptosis on HL-60 cells by FME was mainly associated with ROS production, GSH depletion, mitochondrial dysfunction, and caspase activation.