In this study, a novel exopolysaccharide (EPS) was isolated from the fermentation broth of Trichoderma pseudokoningii and its anticancer activities on human leukemia K562 cells were studied. EPS could significantly inhibited K562 cells proliferation in a time- and concentration-dependent manner. Meanwhile, characteristic of apoptosis, including apoptotic morphological features and the apoptosis rate were obtained. Sequentially, the dissipation of mitochondrial membrane potential, increase production of Reactive oxygen species (ROS), enhancement of the concentration of intracellular, up-regulation of Bax and p53 mRNA, down-regulation of Bcl-2 mRNA were also detected. The results indicate that the EPS could induce of K562 cells apoptosis, primarily in involved the mitochondrial pathways. The present studies suggest that EPS could be a new potential adjuvant chemotherapeutic and chemo preventive agent against human leukemia.