A new lanostanoid ester glucoside, 3 alpha-acetoxy-5 alpha-lanosta-8,24-dien-21-oic acid ester beta-d-glucoside (1), and a known steroid, 2 beta,3 alpha,9 alpha-trihydroxy-5 alpha-ergosta-7,22-diene (2), were isolated from the fruit bodies of Ganoderma tsugae and their structures determined by spectroscopic methods. To study the cytotoxicity of 1 and 2, the changes of DNA content in human hepatocytes (Hep 3B) were studied. A sub-G1 cell stage was drastically increased after 24-h incubation with 1 (24 micrograms/mL). Compound 2 (100 micrograms/mL) inhibited the cell cycle progression of Hep 3B cells at the G2/M phase with an IC50 value of about 87.1 micrograms/mL. These results indicate that 1 causes cell death by apoptosis and 2 may possess the activity of cell cycle inhibition.