Meningitis occurs when blood-borne pathogens cross the blood-brain barrier (BBB) in a complex interplay between endothelial cells and microbial gene products. We sought to understand the initial response of the BBB to the human meningeal pathogen group B Streptococcus (GBS) and the organism’s major virulence factors, the exopolysaccharide capsule and the β-hemolysin/cytolysin toxin (β-h/c). Using oligonucleotide microarrays, we found that GBS infection of human brain microvascular endothelial cells (HBMEC) induced a highly specific and coordinate set of genes including IL-8, Groα, Groβ, IL-6, GM-CSF, myeloid cell leukemia sequence-1 (Mcl-1), and ICAM-1, which act to orchestrate neutrophil recruitment, activation, and enhanced survival. Most strikingly, infection with a GBS strain lacking β-h/c resulted in a marked reduction in expression of genes involved in the immune response, while the unencapsulated strain generally induced similar or greater expression levels for the same subset of genes. Cell-free bacterial supernatants containing β-h/c activity induced IL-8 release, identifying this toxin as a principal provocative factor for BBB activation. These findings were further substantiated in vitro and in vivo. Neutrophil migration across polar HBMEC monolayers was stimulated by GBS and its β-h/c through a process involving IL-8 and ICAM-1. In a murine model of hematogenous meningitis, mice infected with β-h/c mutants exhibited lower mortality and decreased brain bacterial counts compared with mice infected with the corresponding WT GBS strains.