Type I interferons (IFNs) are critical mediators of the innate immune system to defend viral infection. Interferon regulatory factor (IRF) 3 and IRF7 are transcription factors that play critical roles in type I IFN production in response to viral infection. It has been shown that the protein kinase I kappaB kinase alpha (IKK alpha) is critically involved in IRF7 activation and IFN-alpha production in Toll-like receptor 7/9 (TLR7/9) signaling cascades. However, overexpression of IKK alpha does not activate the IFN-alpha promoters. Here we show that the protein kinase nuclear factor kappaB-inducing kinase (NIK) confers IKK alpha the ability to activate IRF3/7. Previous studies have shown that NIK phosphorylates IKK alpha at Ser-176 and Ser-180 residues, and mutation of each of the two residues to glutamate, which mimics its phosphorylation, caused constitutive activation of NF-kappaB. However, mutation of the two serine residues has differential effects on IKK alpha-mediated activation of IRF3/7. While IKK alpha(S176E) constitutively activates IRF3/7, IKK alpha(S180E) losses its ability to activate IRF3/7. These findings suggest that IKK alpha-mediated activation of NF-kappaB and IRF3/7 are differentially regulated by NIK, and NIK plays an important role in TLR7/9-mediated IFN-alpha production.