As a prerequisite for colonization or causing local infections, Streptococcus pyogenes (group A streptococci, GAS) need to specifically adhere to eukaryotic cell surfaces. Predominantly responsible adhesin genes are contained in a genotype-specific pattern within the FCT region of the GAS genome. In this study, MsmR, belonging to AraC/XylS type transcriptional regulators, was identified in the FCT region as a positive regulator of the major fibronectin-binding adhesin protein F2 in a serotype M49 strain. Compared with the wild-type strain, the msmR mutant showed reduced binding to immobilized fibronectin and decreased adherence to and internalization into human pharyngeal epithelial cells. These results suggested that altered levels of fibronectin-binding proteins in the mutant affect eukaryotic cell attachment and internalization. Complete transcriptome and reporter fusion assay data revealed that MsmR positively regulates FCT region genes including Nra and cytolysin-mediated translocation system genes. Consistent with the genetic data, the mutant showed attenuated streptolysin O activity and eukaryotic cell cytotoxity. Direct binding of recombinant MsmR to nga, nra/cpa and prtF2 promoter regions was confirmed by EMSA assays. As prior analysis demonstrated the Nra regulator negatively affects gene expression from the FCT region, MsmR and Nra appear to adversely control crucial virulence factor expression in GAS and thus contribute to a fine-tuned balance between local destructive process and metastatic spreading of the bacteria.