[Objective] To explore the effects of foot-and-mouth disease virus (FMDV) structural protein VP0 in type I IFN signaling.[Methods] A recombinant VP0 protein was constructed and expressed in eukaryotic cells followed by FMDV infection. The influence of VP0 protein during FMDV replication in BHK cells was determined by RT-PCR and the mRNA levels of IFN-β, RIG-I, ISG15, ISG20 and IRF3 was assessed. The dose-dependent effects of VP0 protein on Sendai virus (SeV)-triggered activation of IFN-β and NF-κB promoter as well as on RLRs-mediated activation of IFN-β promoter were examined by luciferase assay. Co-immunoprecipitation was done to detect the interaction of VP0 protein and the components of the RLRs signaling pathway.[Results] The recombinant VP0 protein was successfully expressed in HEK-293T cells and confirmed by Western blotting. At 4-6 h post-infection, VP0 protein significantly promoted the replication of FMDV in BHK cells (P<0.01 or P<0.05). VP0 protein clearly inhibited the gene expression level of IFN-β, RIG-I, ISG15, ISG20 and IRF3 (P<0.01 or P<0.05), respectively. In luciferase assays, FMDV VP0 protein distinctly inhibited SeV-triggered activation of the IFN-β and NF-κB promoters in a dose-dependent manner. The interferon regulatory factor 3 (IRF3)-activated IFN-β promoter and its upstream components, RIG-I, MDA5, VISA and TBK1 were also down-regulated in the presence of VP0 protein. The inhibition of IFN-β promoter induced by IRF7 was not observed. Furthermore, Co-immunoprecipitation showed that VP0 interacted with IRF3 protein in HEK-293T cells.[Conclusion] Our results indicated that VP0 may inhibit the activation of type I IFN signaling pathway via interaction with IRF3.