Protein priming is an important mechanism that many viruses use to initiate genomic DNA or RNA synthesis. Birnaviruses are the only double-stranded (ds) RNA viruses that use protein priming. The viral-encoded VP1 of birnavirus functions as both a polymerase and a protein primer and is able to undergo self-guanylylation to acquire a covalently linked rGMP. By employing biochemical assays using recombinant proteins, we have shown that VP1 self-guanylylation does not require an RNA template but is dependent on divalent metal ions. VP1 reacts with all four types of rNTPs but strongly prefers rGTP. Unexpectedly, two fatal polymerase mutants D402A and E421Y, each having an essential catalytic residue mutated and unable to catalyze RNA synthesis, remain active in self-guanylylation. The guanylylation site was further mapped to the VP1 N-terminal domain. Our results support a mechanism in which VP1 self-guanylylation is catalyzed by a novel active site different from the polymerase active site.