Inactivation of Rho GTPase with a single intraocular injection of Rho antagonists stimulates survival and regeneration of retinal ganglion cells (RGCs) after optic nerve injury. However, this effect is short-lived. Here we tested the impact of multiple injections of C3-like Rho antagonists on RGC viability and axon regeneration after optic nerve lesion. Our data show that both neuronal survival and axon regeneration were enhanced with repeated delivery of cell-permeable C3. We found an approximately 1.5-fold increase in RCG survival when additional Rho antagonist injections were performed after the first week from the time of lesion. In contrast, increased regeneration required early inactivation of Rho and injections performed in the second week did not further enhance regenerative outcome. These results reveal differences in the length of the therapeutic windows through which Rho inactivation acts on RGC survival or regeneration after axotomy.