The sulforhodamine B (SRB) assay was employed to evaluate the anti-tumor effect of petroleum ether extracts from Fomitopsis officinalis (Vill.:Fr.) Bond (PEFO) on SMCC- 7721,SGC-7901,and Hep-2 cell lines in vitro. The results showed PEFO inhibited the growth of three kinds of cells in dose-dependent manner with the IC50 values on SMCC-7721,SGC-7901,and Hep-2 cells being of 58.74,74.13 and 83.18 μg/mL, respectively. The SMCC-7721 cells exposed to PEFO obviously changed cell morphology and cellular ultrastructure. The SMCC-7721 cell presented wrinkled nuclei, poor cell adherent growth and cell lysis, while mitochondria swell and a large number of vacuoles in cytoplasm were observed by transmission electron microscope. The flow cytometry results revealed that SMCC-7721cell decreased in S-phase and increased in G0/G1-phase, which arrested in G0/G1 phase. The study demonstrated that PEFO could inhibit cell proliferation in vitro, and the anti-tumor activity of PEPO might be achieved by inhibition of tumor cellular aerobic metabolism and intervention cell cycle.