In adult mammals, after optic nerve injury, retinal ganglion cells (RGCs) do not regenerate their axons and most of them die by apoptosis within a few days. So far, many molecules injected intravitreally were able to enhance RGCs survival and axonal regeneration, however with poor results. The rho-related small GTPase Rac1 is part of a complex, still not fully understood, intracellular signaling network, mediating in neurons many effects, including axon growth and cell survival. However, its role in neuronal survival and regeneration in vivo has not yet been properly investigated. To address this point we intravitreally injected selective cell-penetrating Rac1 mutants after optic nerve crush and studied the effect on RGCs survival and axonal regeneration in transgenic mice. We injected two well-characterized L61 constitutively active Tat-Rac1 fusion protein mutants, in which a second F37A or Y40C mutation confers selectivity in downstream signaling pathways. Results showed that, 15 days after crush, both mutants were able to improve survival, while the double mutant harboring the F37A mutation also improved the axonal regeneration. Furthermore, we showed that the two mutants exert their effect by distinct mechanisms: the F37A action is related to Pak1 activation on RGCs, whereas the Y40C mutant gave an ERK1/2 activation on glial cells. Thus, the selective activation of specific Rac1-dependent pathways could represent a promising strategy to contrast neuronal degeneration.