The Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that must adapt to unique host environments in order to survive. Links between sugar metabolism and virulence have been demonstrated in GAS, where mutants in the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exhibited Streptolysin S (SLS)-mediated hemolysis during exponential growth. This early onset hemolysis correlated with an increased lesion size and severity in a murine soft tissue infection model when compared with parental M1T1 MGAS5005. To identify the PTS components responsible for this phenotype, we insertionally inactivated the 14 annotated PTS EIIC-encoding genes in the GAS MGAS5005 genome to functionally characterize each EIIC. It was found that a few EIIs had a limited in uence on PTS sugar metabolism, whereas others were promiscuous. The mannose-speci c EII locus exhibited the most in uence on PTS sugar metabolism. Importantly, the mannose-speci c EII also acted to prevent the early onset of SLS-mediated hemolysis. These roles were not identical in two different M1T1 GAS strains, highlighting the versatility of the PTS to adapt to strain-speci c needs. This is further illustrated by the fructose-speci c EII, which is important for survival in whole human blood for MGAS5005, but not 5448. The mannose-speci c EII can transport glucose in other pathogens, but the route of glucose utilization is unknown in GAS. MGAS5005 mutants were generated in a non-PTS glucose transporter (GlcU) and a glucokinase (NagC) of an annotated non-PTS glucose metabolic pathway. Since ∆ptsI, ∆nagC, and ∆glcU all grow to some extent in glucose, it is evident that glucose can be metabolized both by PTS and non-PTS routes. . However, the route of glucose utilization affects overall pathogenesis, as ∆nagC survives like WT in whole human blood, whereas ptsI is unable to survive. Subcutaneous infection of mice with ∆nagC did not exhibit increased lesion size, although these lesions are more severe than MGAS5005 due to the early onset of hemolysis. Overall this suggests that the routes of glucose metabolism greatly in uence SLS-mediated hemolysis. These results highlight that PTS carbohydrate metabolism plays an important role for GAS pathogenesis in both the skin and whole human blood, through the actions of EIIs.