Osteopontin (OPN) is a multifunctional protein involved in both innate immunity and adaptive immunity. However, the function of OPN, especially the intracellular form OPN (iOPN) on innate antiviral immune response remains elusive. Here, we demonstrated that iOPN is an essential positive regulator to protect the host from virus infection. OPN deficiency or knockdown significantly attenuated virus-induced IRF3 activation, IFN-β production and antiviral response. Consistently, OPN-deficient mice were more susceptible to VSV infection than WT mice. Mechanistically, iOPN was found to interact with tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) and inhibit Triad3A-mediated K48-linked polyubiquitination and degradation of TRAF3 through the C-terminal fragment of iOPN. Therefore, our findings delineated a new function for iOPN to act as a positive regulator in innate antiviral immunity through stabilization of TRAF3.