Hypsizigus marmoreus has recently become a popular edible mushroom in Asia. Despite its extensive use, the underlying mechanisms of the anticarcinogenic effects on the initiation stage are not precisely known. Therefore, methanol extracts from H. marmoreus were prepared and then tested for antiproliferative effects in cancer cells and antimutagenic activities as well as mutagenic capacity using the Ames Salmonella mutagenicity test. In addition, the effects on the phase I drug metabolizing enzymes, phase II detoxifying enzymes, and antioxidative activities were evaluated in livers from mice pretreated with methanol extracts from H. marmoreus and challenged with benzo[a]pyrene (B[a]P). In the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, methanol extracts from H. marmoreus displayed a dose-dependent inhibitory effect against human hepatocarcinoma and colon carcinoma cells. However, equivalent doses did not induce mutagenicity when tested with Salmonella typhimurium TA98 and TA100 while exhibiting antimutagenicity against direct-acting and indirect-acting mutagens. Methanol extracts from H. marmoreus strongly decreased total cytochrome P450 and activity of ethoxyresorufin deethylase after B[a]P challenge. Further investigation revealed that methanol extracts from H. marmoreus decreased protein levels of cytochrome P450 IAI isozyme induced by B[a]P. Methanol extracts from H. marmoreus increased the content of glutathione and activity of glutathione S-transferase. This also induced the activity of quinone reductase, an enzyme well known to be anticarcinogenic. The results of the present study therefore demonstrated that methanol extracts from H. marmoreus may have antimutagenic effects, inhibiting the mutagenicity of some mutagens, particularly indirect-acting B[a]P. The mechanism of this antimutagenicity may be the induction of the activity of phase II enzymes, as well as the ability to reduce phase I metabolic-activating enzymes in mouse liver.