The innate immune system is the first line of defense against bacterial and viral infections. The recognition of pathogen-associated molecular patterns by the RIG-I–like receptors, TLRs, and cGAS leads to the induction of IFN-I by activating the transcription factor IRF-3. Although the mechanism of IRF-3 activation has been extensively studied, the structural basis of IRF-3 activation upon phosphorylation is not fully understood. In this study, we determined the crystal structures of phosphorylated human and mouse IRF-3 bound to CREB-binding protein (CBP), which reveal that phosphorylated IRF-3 forms a dimer via pSer 386 (pSer 379 in mouse IRF-3) and a downstream pLxIS motif. Size-exclusion chromatography and cell-based studies show that mutations of key residues interacting with pSer 386 severely impair IRF-3 activation and IFN-β induction. By contrast, phosphorylation of Ser 396 within the pLxIS motif of human IRF-3 only plays a moderate role in IRF-3 activation. The mouse IRF-3/CBP complex structure reveals that the mechanism of mouse IRF-3 activation is similar but distinct from human IRF-3. These structural and functional studies reveal the detailed mechanism of IRF-3 activation upon phosphorylation.