Viperin is an interferon-inducible protein inhibiting many DNA and RNA viruses. It contains an N-terminal transmembrane helix, a highly conserved C-terminus and a middle region carrying a CX3CX2C motif, characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. So far no structural characterization has been reported and reconstitution of the [4Fe–4S] cluster in viperin all failed. Here, by dissecting the 361-residue human viperin into 12 fragments, followed by extensive CD and NMR characterization, Viperin (45–361) was identified to be soluble and structured in buffers. Most importantly, we have successfully reconstituted the [4Fe–4S] cluster in Viperin (45–361), thus providing the first experimental evidence confirming that viperin is indeed a radical SAM enzyme. Furthermore, the C-terminus Viperin (214–361) which is insoluble in buffers but again can be solubilized in salt-free water appears to be only partially folded. Our results thus imply that the radical SAM enzyme activity may play a key role in the broad antiviral actions of viperin.