Fomitopsis pinicola (Sw. Ex Fr. m) Karst (FPK) is a traditional Chinese herb for treatment of fever, analgesic, anti-inflammatory and especially for antitumor, but the inhibitory effect of active compounds on the proliferation of leukemia cells and its potential mechanism remains unclear. The present study was to evaluate the possible anti-proliferative activity of three organic fractions from F. pinicola on three leukemia cells. MTT assay showed that FPK chloroform extract (FPKc) displayed higher anti-tumor effect on U937 cells than that of ethylacetate and butanol fraction. Flow cytometry studies revealed that FPKc induced ROS accumulation in U937 cells. Loss of plasma and mitochondrial membrane potential, increase of intracellular Ca²⁺ concentration and the rate of apoptosis were observed clearly after treatment with FPKc. However, NAC as ROS inhibitor, could significantly alleviate the occurrence of cell apoptosis and improve the ratio of viable cells. Therefore, ROS generated in U937 cells played an important role in F. pinicola induced apptosis.