Liver is suggested to be the major target of dengue virus infection and plays an important role in the immunopathogenesis of dengue hemorrhagic fever. Previously, we reported that five human liver cell lines (HuH-7, HA22T, Hep3B, PLC, and Chang liver) with various degrees of differentiation and tumorigenicity showed different susceptibility for dengue virus infection. Here, we demonstrate that heparin, an analogue of heparan sulfate (HS), can compete with HS on cell membrane for virus binding and subsequently inhibits the replication of dengue-2 and Japanese encephalitis viruses in hepatoma and BHK-21 cells, respectively. It indicates that the binding of these viruses with HS is an important process for their invasion. Moreover, the inhibitory effect of heparin correlates with the infectivity of the virus in the cells. All together, our results suggest that HS is an important host component for dengue and Japanese encephalitis virus replication, which can be effectively blocked by heparin.