Argonaute (Ago) proteins are conserved programmable nucleases present in eukaryotes and prokaryotes and provide defense against mobile genetic elements. Almost all characterized pAgos prefer to cleave DNA targets. Here, we describe a novel pAgo from bacterium (VbAgo) that can specifically cleave RNA targets rather than DNA targets at 37°C and function as a multiple-turnover enzyme showing prominent catalytic capacity. VbAgo utilizes DNA guides (gDNAs) to cleave RNA targets at the canonical cleavage site. Meanwhile, the cleavage activity is remarkably strengthened at low concentrations of NaCl. In addition, VbAgo presents a weak tolerance for mismatches between gDNAs and RNA targets, and single-nucleotide mismatches at positions 11‒12 and dinucleotide mismatches at positions 3‒15 dramatically reduce target cleavage. Moreover, VbAgo can efficiently cleave highly structured RNA targets at 37°C. These properties of VbAgo broaden our understanding of Ago proteins and expand the pAgo-based RNA manipulation toolbox.