The single-celled human parasite Entamoeba histolytica possesses a dynamic actin 
cytoskeleton vital for its intestinal and systemic pathogenicity. The E. 
histolytica genome encodes several Rho family GTPases known to regulate 
cytoskeletal dynamics. EhRho1, the first family member identified, was reported 
to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, 
raising the possibility that it may be a misclassified Ras family member. Here, 
we report the crystal structures of EhRho1 in both active and inactive states. 
EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian 
Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of 
mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin 
reorganization and microtubule formation during mitosis. EhRho1, but not a 
constitutively active mutant, interacts with a newly identified EhRhoGDI in a 
prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces 
actin stress fiber formation in mammalian fibroblasts, thereby identifying it as 
a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide 
exchange relative to mammalian Rho GTPases due to a distinctive switch one 
isoleucine residue reminiscent of the constitutively active F28L mutation in 
human Cdc42, which for the latter protein, is sufficient for cellular 
transformation. Nonconserved, nucleotide-interacting residues within EhRho1, 
revealed by the crystal structure models, were observed to contribute a 
moderating influence on fast spontaneous nucleotide exchange. Collectively, 
these observations indicate that EhRho1 is a bona fide member of the Rho GTPase 
family, albeit with unique structural and functional aspects compared with 
mammalian Rho GTPases.