The production of cytokines such as type I interferon (IFN) is an essential component of innate immunity. Insufficient amounts of cytokines lead to host sensitivity to infection, whereas abundant cytokine production can lead to inflammation. A tight regulation of cytokine production is, thus, essential for homeostasis of the immune system. IFN-α production during RNA virus infection is mediated by the master transcription factor IRF7, which is activated upon ubiquitination by TRAF6 and phosphorylation by IKKε and TBK1 kinases. We found that Fas-associated death domain (FADD), first described as an apoptotic protein, is involved in regulating IFN-α production through a novel interaction with TRIM21. TRIM21 is a member of a large family of proteins that can impart ubiquitin modification onto its cellular targets. The interaction between FADD and TRIM21 enhances TRIM21 ubiquitin ligase activity, and together they cooperatively repress IFN-α activation in Sendai virus-infected cells. FADD and TRIM21 can directly ubiquitinate IRF7, affect its phosphorylation status, and interfere with the ubiquitin ligase activity of TRAF6. Conversely, a reduction of FADD and TRIM21 levels leads to higher IFN-α induction, IRF7 phosphorylation, and lower titers of RNA virus of infected cells. We conclude that FADD and TRIM21 together negatively regulate the late IFN-α pathway in response to viral infection.