Viral infection activates transcription factors IRF3 and NF-κB, which collaborate to induce type I interferons (IFNs). Here, we identified glycogen synthase kinase 3β (GSK3β) as an important regulator for virus-triggered IRF3 and NF-κB activation, IFN-β induction, and cellular antiviral response. Overexpression of GSK3β potentiated virus-induced activation of IRF3 and transcription of the IFNB1 gene, whereas reduced expression or deletion of GSK3β impaired virus-induced IRF3 and NF-κB activation, transcription of the IFNB1 gene, as well as cellular antiviral response. GSK3β physically associated with the kinase TBK1 in a viral infection-dependent manner. GSK3β promoted TBK1 self-association and autophosphorylation at Ser172, which is critical for virus-induced IRF3 activation and IFN-β induction. The effect of GSK3β on virus-induced signaling is independent of its kinase activity. Our findings suggest that GSK3β plays important roles in virus-triggered IRF3 activation by promoting TBK1 activation and provide new insights to the molecular mechanisms of cellular antiviral response.