We show here that the encoded proteins of the two duplicated RHO1 genes from the filamentous fungus Ashbya gossypii, AgRHO1a and AgRHO1b have functionally diverged by unusual mutation of the conserved switch I region. Interaction studies and in vitro assays suggest that a different regulation by the two GTPase activating proteins (GAPs) AgLrg1 and AgSac7 contributes to the functional differences. GAP-specificity and protein function is determined to a large part by a single position in the switch I region of the two Rho1 proteins. In AgRho1b, this residue is a tyrosine that is conserved among the Rho-protein family, whereas AgRho1a carries an atypical histidine at the same position. Mutation of this histidine to a tyrosine changes GAP-specificity, protein function and localization of AgRho1a. Furthermore, it enables the mutated allele to complement the lethality of an AgRHO1b deletion. In summary, our findings show that a simple mutation in the switch I region of a GTP-binding protein can change its affinity towards its GAPs, which finally leads to a decoupling of very similar protein function without impairing effector interaction.