Streptococcus pyogenes, or Group A Streptococcus (GAS), is a human pathogen that causes a wide range of diseases, including pharyngitis, necrotizing fasciitis and toxic shock syndrome. This bacterium produces a large arsenal of virulence factors, including the cell wall-anchored Streptococcus pyogenes nuclease A (SpnA), which facilitates immune evasion by degrading the DNA backbone of neutrophil extracellular traps. SpnA consists of a C-terminal endo/exonuclease domain and a N-terminal domain of unknown function. Site-directed mutagenesis of SpnA has been carried out to further define the mechanism of the nuclease. The ability to degrade DNA by these recombinant SpnA mutants was either abolished or reduced when predicted metal-binding and catalytic site residues were mutated. To investigate the role of SpnA in virulence in vivo, Galleria mellonella (wax moth) larvae were used as an infection model. A GAS spnA deletion mutant showed reduced virulence in this model, with the spnA wt complementation completely restoring virulence. Interestingly, complementation with the spnA catalytic site mutant SpnA H716A only partially restored virulence. Our results outline the critical role of several predicted residues in enzymatic activity and demonstrate that nuclease activity is not exclusively responsible for SpnA-mediated GAS virulence in a Galleria mellonella infection model.