Contradictory results have been reported regarding NS3 in RNA synthesis. To investigate the effect of NS3 on classical swine fever virus (CSFV) NS5B RNA-dependent RNA polymerase activity (RdRp) activity and NS3-NS5B interaction, RdRp reactions, GST-pull-down assays and co-immunoprecipitation analyses containing NS5B and either of NS3 protein and the different truncated NS3 mutants were performed, respectively. We found that NS3 stimulated NS5B RdRp activity in a dose-dependent manner by binding to NS5 through a NS3 protease domain. Furthermore, mapping important regions of the NS3 protease domain was carried out by deletion mutagenesis, associated with RdRp reactions, GST-pull-down assays and co-immunoprecipitation analyses. Results showed that stimulation of CSFV NS5B RdRp activity was obtained by NS3 binding to NS5B through a 31-amino acid fragment at the N-terminal end of NS3 protease domain, which mediated a specific NS3-NS5B interaction.