Objective To compare the antitumor effects and chemical components of the extracts in cultivated Cordyceps sinensis (Cs) fungus HK-1 and natural Cs. Methods The cultivated Cs fungus HK-1 and natural Cs were extracted with petroleum ether (PE), ethyl acetate (EtOAc), ethanol (EtOH), and water. The cytotoxicity of all the extracts was observed with MTT assay on breast cancer cell line MCF-7, mouse melanoma cell line B16, human premyelocytic leukemia cell line HL-60, and human hepatocellular carcinoma cell line Hep G2. The antitumor activity in vivo of the extract was further studied with animal model. The chemical constituents in the extracts were analyzed by HPLC. Results All of the extracts from the cultivated Cs fungus HK-1 had much stronger cytotoxicity on B16 than those from natural Cs, and the EtOAc extract had the most potent activity. The activity of EtOAc extract from the cultivated Cs fungus HK-1 was more potent than that from natural Cs on all the four cancer cell lines. EtOAc extract from the cultivated Cs fungus HK-1 also can inhibit the growth of tumor in vivo. Chemical components of all extracts were analyzed and ergosterol and adenosine were found to be potential compounds. Conclusion The cultivated Cs fungus HK-1 has stronger antitumor activity than natural Cs and is a potential source of antitumor compounds.