Hepatitis C virus (HCV) is a hepatotropic single-stranded RNA virus. HCV infection is causally linked with development of liver cirrhosis and hepatocellular carcinoma. Enhanced production of reactive oxygen species by HCV has been implicated to play an important role in HCV-induced pathogenesis. Mangosteen has been widely used as a traditional medicine as well as a dietary supplement ,thanks to its powerful anti-oxidant effect. In the present study, we demonstrated that the ethanol extract from mangosteen fruit peels (MG-EtOH) is able to block HCV genome replication using HCV genotype 1b Bart79I subgenomic (EC50 5.1 μg/mL) and genotype 2a J6/JFH-1 infectious replicon systems (EC50 3.8 μg/mL). We found that inhibition of HCV replication by MG-EtOH led to subsequent down-regulation of expression of HCV proteins. Interestingly, MG-EtOH exhibited a modest inhibitory effect on in vitro RNA polymerase activity of NS5B. Among a number of xanthones compounds identified within this MG-EtOH, we discovered α-MG (EC50 6.3 μM) and γ-MG (EC50 2.7 μM) as two major single molecules responsible for suppression of HCV replication. This finding will provide a valuable molecular basis to further develop mangosteen as an important dietary supplement to combat HCV-induced liver diseases.