Dengue fever has become a global concern as dengue infections are prevalent in the tropics and the subtropics, and can lead to a severe life-threatening illness. Currently, there is a widespread interest to search for vaccine or antiviral therapy to combat DENV infection. In this study, we have identified emetine dihydrochloride with potent antiviral activity against DENV infection. Emetine was also shown to inhibit DENV replication consistently in all the dengue serotypes. Experiments were designed to define the stage of viral replication cycle at which emetine blocked DENV infection. Emetine did not target the entry process of DENV into cells as inhibitory effect of emetine on DENV infectivity remained when naked dengue viral RNA were transfected directly into the cells. Thus, we further investigated the inhibitory effect of emetine through timecourse studies and emetine was shown to strongly inhibit DENV infection at early stage of viral replication cycle by either affecting the synthesis viral RNA pathway or viral protein translation pathway. Quantitative RT-PCR assay indicated that emetine strongly reduced the production of positive-strand and negative-strand of DENV RNA. Ultrastructural analysis of emetine-treated cells further revealed that the formations of membranous replication complexes of DENV within cells were aborted in the presence of emetine. Together, these results suggest that emetine can inhibit DENV infection by impeding viral RNA synthesis therefore emetine could be further assessed and developed as a potential antiviral therapeutic agent against DENV infection.