The hepatitis delta virus ribozyme catalyzes an RNA cleavage reaction using a catalytic nucleobase and a divalent metal ion. The catalytic base, C75, serves as a general acid and has a pKa shifted towards neutrality. Less is known about the role of metal ions in the mechanism. A recent crystal structure of the pre-cleavage ribozyme identified a Mg2+ ion that interacts through its partial hydration sphere with the G25•U20 reverse wobble. In addition, this Mg2+ ion is in position to directly coordinate the nucleophile, the 2’-hydroxyl of U(-1), suggesting it can serve as a Lewis acid to facilitate deprotonation of the 2’-hydroxyl. To test the role of the active site Mg2+ ion, we replaced the G25•U20 reverse wobble with an isosteric A25•C20 reverse wobble. This change was found to significantly reduce the negative potential at the active site, as supported by electrostatics calculations, suggesting that active site Mg2+ binding could be adversely affected by the mutation. Kinetic analysis and molecular dynamics of the A25•C20 double mutant suggest that this variant stably folds into an active structure. However, pH-rate profiles of the double mutant are inverted relative to the profiles for wild-type ribozyme, suggesting that the A25•C20 double mutant has lost the active site metal ion. Overall, these studies support a model wherein the partially hydrated Mg2+ positioned at the G25•U20 reverse wobble is catalytic and could serve as a Lewis acid, a Brønsted base, or both to facilitate deprotonation of the nucleophile.