Here we have investigated the function of TcRho1, a Rho family orthologue from the parasite Trypanosoma cruzi. We have selected parasites overexpressing wild-type TcRho1 and a truncated form of TcRho1 (TcRho1-DeltaCaaX) which is unable to undergo farnesylation and supposed to interfere with recruitment of Rho effectors to membranes. TcRho1 protein was localized at the anterior region of wild-type and TcRho1 overexpressing epimastigotes, suggesting association with the Golgi apparatus. Accordingly, parasites overexpressing TcRho1-DeltaCaaX presented cytoplasmic fluorescence. To address the function of TcRho1 during differentiation, from epimastigotes to trypomastigotes, we submitted parasites overexpressing the above-cited lineages to metacyclogenesis assays. Parasites overexpressing TcRho1-DeltaCaaX generated a discrete number of metacyclic trypomastigotes when compared with other lineages. Strikingly, TcRho1-DeltaCaaX cells died synchronously during the process of metacyclogenesis.