Calycosin-7-O-beta-D-glucopyranoside (CCGR) is the main isoflavonoid compound isolated from Astragalus membranaceus var. Mongholicu (BGE.) HSIAO, a Chinese herb medicine traditionally used to treat viral myocarditis. In this study, its antiviral activities against coxsackie virus B3 (CVB3) causing myocarditis were investigated. In vitro assay showed that CCGR displayed a low cytotoxicity and effectively inhibited CVB3-mediated cytopathic effects on Vero cells with an IC(50) value of 25 microg/ml. In an acute myocarditis murine model, treatment with 24 mg/kg CCGR for 14 d significantly improved the survival rate of mice infected with CVB3, alleviated pathological damages of cardiac muscles in the myocarditis mice, reduced the virus titers in the heart, decreased heart indexes and improved left ventricular function. These results showed that CCGR exerted significant antiviral activities against CVB3 both in vitro and in vivo, and identified CCGR as one of active ingredients in Astragalus membranaceus for the treatment of viral myocarditis.