This study examined the anti-viral effect of ursolic acid on guinea pig cytomegalovirus (GPCMV) and explored the steps of viral replication targeted by ursolic acid. Cytopathic effect assay and MTT method were employed to determine the 50% cellular cytotoxicity (CC50), 50% effective concentration (EC50) and therapeutic index (TI) with GPCMV. To investigate the specific anti-viral effect of ursolic acid at different temperatures and time points, two other medicines, ganciclovir and Jinyebaidu (JYBD), serving as controls, were studied for comparison. Our results showed that the CC50 of ganciclovir, JYBD and ursolic acid were 333.8, 3015.6, 86.7 mu g/mL, respectively; EC50 of ganciclovir, JYBD and ursolic acid was 48.1, 325.5 and 6.8 mu g/mL, respectively; TI of ganciclovir, JYBD and ursolic acid was 7, 9, 13, respectively. Similar with ganciclovir, ursolic acid could inhibit the viral synthesis, but did not affect the viral adsorption onto and penetration into cells. We are led to conclude that the anti-cytomegalovirus effect of ursolic acid is significantly stronger than ganciclovir or JYBD, and the cytotoxic effect of ursolic acid lies in its ability to inhibit viral synthesis.