In this study, we investigated the specific inhibition of West Nile virus (WNV) NS2B-NS3 protease and viral propagation by palmatine, a chemical compound from Coptis chinensis Franch. It was demonstrated that palmatine could inhibit WNV NS2B-NS3 protease activity in an uncompetitive manner, with a 50% inhibitory concentration (IC(50)) of 96 microM. Palmatine suppressed WNV without detectable cytotoxicity (a 50% effective concentration [EC(50)] of 3.6 microM and a 50% cytotoxicity concentration [CC(50)] of 1,031 microM). Furthermore, palmatine could also suppress dengue virus and yellow fever virus in a dose-dependent manner. This compound could potentially be developed for the treatment of flavivirus infections.