Streptococcus agalactiae, also, known as Group B Streptococcus (GBS) is an important cause of invasive disease, in newborns and certain adult population such as the elderly and pregnant women. The development of GBS disease is initiated by the asyptomatic colonization of the female genital tract and during pregnancy can be associated with chorioamnionnitis, puerperal endometritis and preterm labor. The gastrointestinal and vaginal tracts harbor GBS. This research focuses on understanding the GBS molecular determinants responsible for GBS colonization and niche establishment as well as the role of vaginal microbiota in this process. The first aim is to study the interaction of GBS with human vaginal epithelium and in relation with native vaginal organisms, namely, Lactobacillus spp. Also, we wish to identify the genes that might be involved in these aforementioned interactions. Secondly, we seek to characterize colonization of the vaginal ecosystem by GBS in vivo and study the persistence of GBS in a newly developed murine model. Using our in vitro model we identified that GBS is able to adhere to vaginal epithelium, and is not displaced but is outcompeted and excluded at higher concentrations of Lactobacilli. Also, we studied a two component regulating system, CovR/S might play a role in vaginal establishment of GBS. Using our new in vivo model of GBS infection, we studied the establishment of GBS in the vaginal vault and its interaction with native microflora. These studies will provide fundamental insights into the nature of GBS colonization, which is the critical first step preceding neonatal transmission, infection, and disease.