Recent reaction intermediates have detailed how nucleic acid hydrolysis occurs in the RNA ribonuclease H1 (RNase H1), a fundamental metalloenzyme involved in maintaining the human genome. At odds with the previous characterization, these structures unexpectedly captured multiple metal ions (K and Mg) transiently bound in the vicinity of the two-metal-ion active site. Using multi-microsecond all-atom molecular dynamics and free-energy simulations, we investigated the functional implications of the dynamic exchange of multiple K and Mg ions at the RNase H1 reaction center. We found that such ions are timely positioned at non-overlapping locations near the active site, at different stages of catalysis, being crucial for both reactants' alignment and leaving group departure. We also found that this cation trafficking is tightly regulated by variations of the solution's ionic strength and is aided by two conserved second-shell residues, E188 and K196, suggesting a mechanism for the cations' recruitment during catalysis. These results indicate that controlled trafficking of multi-cation dynamics, opportunely prompted by second-shell residues, is functionally essential to the complex enzymatic machinery of the RNase H1. These findings revise the current knowledge on the RNase H1 catalysis and open new catalytic possibilities for other similar metalloenzymes including, but not limited to, CRISPR-Cas9, group II intron ribozyme and the human spliceosome.