Streptococcus agalactiae is a major neonatal pathogen that is able to colonise various host environments and is associated with both gastrointestinal and vaginal maternal carriage. Maternal vaginal carriage represents the major source for transmission of S. agalactiae to the foetus/neonate and thus is a significant risk factor for neonatal disease. In order to understand factors influencing maternal carriage we have investigated growth and long term survival of S. agalactiae under conditions of low pH and nutrient stress in vitro. Surprisingly, given that vaginal pH is normally <4.5, S. agalactiae was found to survive poorly at low pH and failed to grow at pH 4.3. However, biofilm growth, although also reduced at low pH, was shown to enhance survival of S. agalactiae. Proteomic analysis identified 26 proteins that were more abundant under nutrient stress conditions (extended stationary phase), including a RelE family protein, a universal stress protein family member and four proteins that belong to the Gls24 (PF03780) stress protein family. Cumulatively, these data indicate that novel mechanisms are likely to operate that allow S. agalactiae survival at low pH and under nutrient stress during maternal vaginal colonisation and/or that the bacteria may access a more favourable microenvironment at the vaginal mucosa. As current in vitro models for S. agalactiae growth appear unsatisfactory, novel methods need to be developed to study streptococcal colonisation under physiologically-relevant conditions.