Streptococcus pyogenes (or Group A Streptococcus, GAS) presents a frequent cause of mild to life-threatening infections. The Group A Carbohydrate (GAC) is a conserved surface glycan and the target of GAS rapid-test assays used in clinical diagnostics. GAC exists as three different variants or glycoforms: linear polyrhamnose, polyrhamnose with GlcNAc modifications, and a fully mature glycoform where ~25% of GlcNAc moieties are modified with glycerolphosphate (GroP). Several GAS vaccines currently in clinical development include either the polyrhamnose or GlcNAc-containing variant but not the GroP-modified structure. Since the presence of GroP affects susceptibility to innate host defenses (sPLA2-IIA and Zn2+ toxicity), we aimed to investigate how GroP modifications impacted antibody recognition of GAC. Using a multiplexed bead-based flow cytometric assay, we analyzed systemic antibody responses (IgM, IgG1-3 and IgA) against the three GAC glycoforms in human plasma using fully-defined synthetic structures. We observed the universal presence of GAC-specific IgM and IgG antibodies in plasma of healthy individuals (n=16) and GAS bacteremia patients (n=13). Antibody reactivity was highest for the polyrhamnose-GlcNAc epitope and the presence of GroP significantly decreased IgM and IgG antibody binding to GAC-GlcNAc in both groups. We used the synthetic structures to isolate GAC-specific human B cells and produced recombinant monoclonal antibodies with different epitope specificities as confirmed by binding to fully-defined synthetic structures. Here, we provide insight in the human glycoform-specific antibody repertoire against GAC, an important vaccine antigen. Our findings suggest that the presence of GAC-GroP interferes with antibody recognition, which may affect GAC-related vaccine efficacy.