Group B Streptococcus (GBS) is a normal constituent of the intestinal and vaginal flora in 15–30% of healthy adults. The bacterium can, however, cause infections in elderly and immunocompromised patients, and pregnant women, and is the main cause of fatal invasive disease in newborns. GBS translocation through epithelial, endothelial, and placental barriers is facilitated by a pore-forming toxin β-hemolysin, also known as the β-hemolytic pigmented lipid or granadaene. To identify factors involved in the regulation of β-hemolysin expression we developed new peptide-free culture media for screening of a transposon mutant library of GBS CJB111. The pilot screens identified nine novel genetic loci associated with positive and negative modulation of β-hemolysin expression. Based on preliminary findings, we hypothesize that GBS possesses a regulatory mechanism that tailors β-hemolysin expression to the environment. To unravel the mechanism, we will employ transposon mutant library screening, RNA-seq and metabolomics analyses, isolation, and de novo sequence determination of secreted peptide-based repressors. The results of this exploratory work have the potential to lead to the development of a novel inhibitor of the invasion state of GBS.