Most RNA-cleaving DNAzymes require a metal ion to interact with the scissile phosphate for activity. Therefore, few unmodified DNAzymes work with thiophilic metals because of their low affinity for phosphate. Recently, an Ag+-specific Ag10c DNAzyme was reported via in vitro selection. Herein, Ag10c is characterized to rationalize the role of the strongly thiophilic Ag+. Systematic mutation studies indicate that Ag10c is a highly conserved DNAzyme and its Ag+ binding is unrelated to C-Ag+-C interaction. Its activity is enhanced by increasing Na+ concentrations in buffer. At the same metal concentration, activity decreases in the following order: Li+ > Na+ > K+. Ag10c binds one Na+ ion and two Ag+ ions for catalysis. The pH-rate profile has a slope of ∼1, indicating a single deprotonation step. Phosphorothioate substitution at the scissile phosphate suggests that Na+ interacts with the pro-Rp oxygen of the phosphate, and dimethyl sulfate footprinting indicates that the DNAzyme loop is a silver aptamer binding two Ag+ ions. Therefore, Ag+ exerts its function allosterically, while the scissile phosphate interacts with Na+, Li+, Na+, or Mg2+. This work suggests the possibility of isolating thiophilic metal aptamers based on DNAzyme selection, and it also demonstrates a new Ag+ aptamer.