Drugs targeting 26S proteasome as antitumor agents are considered to be important for cancer therapy. Although the active components are yet to be identified, for more than 1000 years, the low-toxicity Panax ginseng has been used in traditional herbal medicine for either treating or preventing cancer. Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 are distinct components that can be isolated from P. ginseng C.A. Meyer. In this study 26S proteasome was purified from pig red blood cells, and the activity of the seven isolated ginsenosides was analyzed by proteolysis assay. It was found that ginsenoside Rd inhibited 52.9% the chymotrypsin-like activity of 26S proteasome with an IC(50) value of 107.5 microM when Suc-LLVY-AMC was used as a substrate. Ginsenoside Rd displayed a mixed type inhibition of 26S proteasome when analyzed by Lineweaver-Burk plots of the inhibition kinetics. Unlike ginsenoside Rd, the other ginsenosides showed low inhibitory effect of the chymotrypsin-like activity of 26S proteasome. Seven ginsenosides did not inhibit the trypsin-like and caspase-like activities of 26S when Ac-RLR-AMC or Z-LLE-AMC was used as substrate. These results suggest that ginsenoside Rd is a potential drug for cancer prevention due to its specific 26S proteasome inhibitory effect and known low toxicity. Furthermore, both 3-O-Glc(2)-Glc and 20-O-beta-Glc positions of the ginsenoside may play a role in the inhibitory property of the chymotrypsin-like activity in 26S proteasome.