Group B Streptococcus (GBS) colonisation in pregnant women poses a risk of transmission to infants during childbirth, leading to invasive diseases. GBS or Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH), encoded by gapC, is a glycolytic enzyme associated with virulence and immune-mediated protection. However, the role of GAPDH in bacterial phagocytosis and colonisation of the female reproductive tract, a central host niche, is unknown. We generated an S. agalactiae mutant that over-expresses GAPDH (874391p gapC) from gapC using a constitutively active promoter and analysed the mutant in murine macrophage antibiotic protection assays and in virulence assays in vivo, using a colonisation model that is based on experimental infection of the reproductive tract in female mice. Over-expression of GAPDH in GBS did not significantly affect measures of phagocytic uptake compared to an empty vector control. In contrast, 874391pgapC showed a small but statistically significant attenuation for persistence in the reproductive tract of female mice during the chronic phase of infection (10-28 days post-inoculation) relative to the vector control. We conclude that GBS GAPDH elicits the production of multiple cytokines from human cells, and over-expression of GAPDH renders the bacterium more susceptible to host clearance in the female reproductive tract. [...]