The signaling adaptor TRAF3 is a highly versatile regulator of both innate immunity and adaptive immunity, but how its phosphorylation is regulated is still unknown. Here we report that deficiency in or inhibition of the conserved serine-threonine kinase CK1 epsilon suppressed the production of type I interferon in response to viral infection. CK1 epsilon interacted with and phosphorylated TRAF3 at Ser349, which thereby promoted the Lys63 (K63)-linked ubiquitination of TRAF3 and subsequent recruitment of the kinase TBK1 to TRAF3. Consequently, CK1 epsilon-deficient mice were more susceptible to viral infection. Our findings establish CK1 epsilon as a regulator of antiviral innate immune responses and indicate a novel mechanism of immunoregulation that involves CK1 epsilon-mediated phosphorylation of TRAF3.