Type III group B streptococcus (GBS) has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, but the underlying mechanisms remain incompletely understood. In the present study, we showed that the geranylgeranyl transferase I inhibitor, GGTI-298, not the farnesyltransferase inhibitor, FTI-277 inhibited type III GBS invasion of HBMEC. The substrates for GGTI-298 include Rho family GTPases, and we showed that RhoA and Rac1 are involved in type III GBS invasion of HBMEC. This was shown by the demonstration that infection with type III GBS strain K79 increased the levels of activated RhoA and Rac1 and GBS invasion was inhibited in HBMEC expressing dominant-negative RhoA and Rac1. Of interest, the level of activated Rac1 in response to type III GBS was decreased in HBMEC expressing dominant-negative RhoA, while the level of activated RhoA was not affected by dominant-negative Rac1. These findings indicate for the first time that activation of geranylgeranylated proteins including RhoA and Rac1 is involved in type III GBS invasion of HBMEC and RhoA is upstream of Rac1 in GBS invasion of HBMEC.