<span class="paragraphSection"><div class="boxTitle">Abstract</div>Xrn1 is a highly conserved 5′→3′ exoribonuclease that plays a central role in RNA turnover and quality control in eukaryotic cells. Although Xrn1 is known to degrade single-stranded RNA in a processive manner, the mechanism by which it engages and unwinds structured RNA remains incompletely understood. Here, we identify two evolutionarily conserved arginine residues, R100 and R101, located proximal to the active site, as critical determinants of duplex unwinding. Charge-conserving substitutions of these residues with lysine (R100K and R101K) markedly impair Xrn1’s exonuclease activity, with R101K exhibiting a more severe functional defect. These effects are particularly pronounced on structured substrates, including RNA–DNA hybrids, implicating the local electrostatic environment in facilitating duplex destabilization via tight gripping 5′ overhangs. Single-molecule Förster resonance energy transfer measurements reveal that Xrn1 unwinds duplexes in discrete steps, each corresponding to the melting of ~8–9 base pairs. Together, these findings uncover a charge-dependent mechanism of RNA duplex unwinding and establish distinct roles for conserved active site residues in modulating Xrn1’s processivity on structured substrates.</span>