Abstract: To investigate the antitumor activity induced by Ganoderma lucidum spore oil on different tumor cells. Human hepatoma cells(HepG2), human non-small cells lung cancer cells(A549)and human colon cancer cells(HCT116)were selected and tested. Cell viability was determined by MTT assay. Western blot analysis was performed to measure the expression of NF-κB and Caspase-3 activity in order to elucidate the mechanism of apoptotic activity caused by Ganoderma lucidum spore oil and to screen out the most sensitive cancer cell lines to Ganoderma lucidum spore oil. MTT assay demonstrated that Ganoderma lucidum spore oil had a strong inhibitory effect on the growth of three cancer cell lines. Among these cells, A549 cells were most sensitive to Ganoderma lucidum spore oil, followed by HepG2 cells and then by HCT116 cells. The results of Western blot showed that Ganoderma lucidum spore oil could promote the activation of NF-κB pathway, and that the activation of NF-κB signaling pathway in cancer cells treated by Ganoderma lucidum spore oil was stronger in A549 cells, HepG2 cells, HCT116 cells respectively. The detection of Caspase-3 activity showed that ganoderma spore oil could activate Caspase-3 dependent apoptosis pathways, which was more important in A549 cells, HepG2 cells and HCT116 cells. This study found that Ganoderma lucidum spore oil had inhibitory effects on A549, HepG2 and HCT116 cells growth and that its antitumor activity was in time-dose dependence. The mechanism may be related to the activation of NF-κB pathway and the Caspase-3 apoptotic pathway, which could accelerate apoptosis and necrosis of tumor cells. Among the three kinds of cancer cells, A549 cells was most sensitive to Ganoderma lucidum spore oil, followed by the HepG2 cells, and then by HCT116 cells.