Metal-dependent DNA and RNA nucleases are enzymes that cleave nucleic acids with great efficiency and precision. These enzyme-mediated hydrolytic reactions are fundamental for the replication, repair, and storage of genetic information within the cell. Here, extensive classical and quantum-based free-energy molecular simulations show that a cation-pi interaction is transiently formed in situ at the metal core of Bacteriophage-lambda Exonuclease (Exo-lambda), during catalysis. This noncovalent interaction (Lys131-Tyr154) triggers nucleophile activation for nucleotide excision. Then, our simulations also show the oscillatory dynamics and swinging of the newly formed cation-pi dyad, whose conformational change may favor proton release from the cationic Lys131 to the bulk solution, thus restoring the precatalytic protonation state in Exo-lambda. Altogether, we report on the novel mechanistic character of cation-pi interactions for catalysis. Structural and bioinformatic analyses support that flexible orientation and transient formation of mobile cation-pi interactions may represent a common catalytic strategy to promote nucleic acid hydrolysis in DNA and RNA nucleases.