Group B Streptococcus (GBS) is major cause of invasive disease in newborn infants and the leading cause of neonatal meningitis. To gain access to the central nervous system (CNS), GBS must not only subvert host defenses in the bloodstream but also invade and survive within brain microvascular endothelial cells (BMEC), the principal cell layer composing the blood-brain barrier (BBB). While several GBS determinants that contribute to the invasion of BMEC have been identified, little is known about the GBS factors that are required for intracellular survival and ultimate disease progression. In this study we sought to identify these factors by screening a random GBS mutant library in an in vitro survival assay. One mutant was identified which contained a disruption in a two-component regulatory system homologous to CiaR/CiaH, which is present in other streptococcal pathogens. Deletion of the putative response regulator, ciaR, in GBS resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages, and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme, and reactive oxygen species. Furthermore, competition experiments with mice showed that wild-type GBS had a significant survival advantage over the GBS DeltaciaR mutant in the bloodstream and brain. Microarray analysis comparing gene expression between wild-type and DeltaciaR mutant GBS bacteria revealed several CiaR-regulated genes that may contribute to stress tolerance and the subversion of host defenses by GBS. Our results identify the GBS CiaR response regulator as a crucial factor in GBS intracellular survival and invasive disease pathogenesis.