Viperin is a radical S-adenosylmethionine (SAM) enzyme that plays a multifaceted role in the cellular antiviral response; however, the mechanism by which viperin mediates its antiviral effects remains unknown. Viperin is implicated regulating K63-linked poly-ubiquitination of interleukin-1 receptor-associated kinase-1 (IRAK1) by the E3 ubiquitin ligase TNF Receptor-Associated Factor 6 (TRAF6) as part of the Toll-like receptor-7 and 9 (TLR7/9) innate immune signaling pathways. We show that poly-ubiquitination of IRAK1 requires the association of viperin with IRAK1 and TRAF6 and is SAM-dependent. Furthermore, IRAK1 and TRAF6 activate viperin to reductively cleave SAM in the presence of either ATP or CTP. Based on these and other observations, we propose a mechanism in which SAM cleavage switches the viperin-IRAK1-TRAF6 complex between poly-ubiquitination-active and inactive states, thereby regulating TLR7/9 signaling. This finding represents a new regulatory mechanism for protein post-translational modifications by radical SAM enzymes that may also explain viperin's seemingly unconnected antiviral effects.