Ribonuclease L (RNase L) is a latent endoribonuclease in an evolutionarily ancient interferon-regulated dsRNA-activated antiviral pathway. 2'-5' oligoadenylate (2-5A), the product of dsRNA-activated oligoadenylate synthetases (OASes), binds to ankyrin repeats near the amino terminus of RNase L, initiating a series of conformational changes that result in the activation of the endoribonuclease. A phylogenetically conserved RNA structure within group C enteroviruses inhibits the endoribonuclease activity of RNase L. In this study we report the mechanism by which group C enterovirus RNA inhibits RNase L. Viral RNA did not affect 2-5A binding to RNase L. Rather, the viral RNA inhibited the endoribonuclease domain. We used purified RNase L, purified 2-5A, and an RNA substrate with a 5' fluorophore and 3' quencher in FRET assays to measure inhibition of RNase L activity by the viral RNA. The group C enterovirus RNA was a competitive inhibitor of the endoribonuclease with a K(i) of 34 nM. Consistent with the kinetic profile of a competitive inhibitor, the viral RNA inhibited the constitutively active endoribonuclease domain of RNase L. We call this viral RNA the RNase L competitive inhibitor RNA (RNase L ciRNA).