The human protease plasmin plays a crucial role in the capacity of the group A streptococcus (GAS; Streptococus pyogenes) to initiate invasive disease. The GAS strain NS88.2 was isolated from a case of bacteremia from the Northern Territory of Australia, a region with high rates of GAS invasive disease. Mutagenesis of the NS88.2 plasminogen binding M protein Prp was undertaken to examine the contribution of plasminogen binding and cell surface plasmin acquisition to virulence. The isogenic mutant NS88.2prp was engineered whereby four amino acid residues critical for plasminogen binding were converted to alanine codons in the GAS genome sequence. The mutated residues were reverse complemented to the wild-type sequence to construct GAS strain NS88.2prpRC. In comparison to NS88.2 and NS88.2prpRC, the NS88.2prp mutant exhibited significantly reduced ability to bind human plasminogen and accumulate cell surface plasmin activity during growth in human plasma. Utilizing a humanized plasminogen mouse model of invasive infection, we demonstrate that the capacity to bind plasminogen and accumulate surface plasmin activity plays an essential role in GAS virulence.—Sanderson-Smith, M. L., Dinkla, K., Cole, J. N., Cork, A. J., Maamary, P. G., McArthur, J. D., Chhatwal, G. S., Walker, M. J. M protein-mediated plasminogen binding is essential for the virulence of an invasive Streptococcus pyogenes isolate.