Abstract Abortive regeneration in the adult mammalian central nervous system (CNS) is partially mediated through CNS myelin proteins, among which Nogo-A plays an important role. Nogo-66, which is located at the C-terminus of Nogo-A, inhibits axonal regrowth through the Nogo-66/NgR signalling pathway. In this study, two small peptides were tested in a neurite outgrowth assay and spinal cord injury (SCI) model to examine the effects of these molecules on the inhibition of Nogo-66/NgR signalling. PepIV was selected from a phage display peptide library as a Nogo-66 binding molecule. And PepII was synthesized as a potential NgR antagonist. The results indicated that PepIV and PepII decrease the mRNA levels of the small GTPase RhoA and partially neutralize CNS myelin inhibition to cultured cerebellar granule cells (CGCs). Moreover, treatment with both peptides was propitious to maintaining residual axons after SCI, thereby promoting regeneration and locomotion recovery. Because RhoA plays a role in stabilizing the cytoskeleton in growth cones and axons, enhanced neurite outgrowth might reflect a decrease in RhoA expression through PepIV and PepII treatment. Moreover, PepIV induced lower RhoA mRNA expression compared with PepII. Therefore, PepIV could block Nogo-66/NgR signalling and reduce RhoA mRNA level, and then contribute to neuronal survival and axonal regrowth after SCI, showing its ability to reverse CNS myelin inhibition to regeneration. Furthermore, selected small peptide might cover some unknown active sites on CNS myelin proteins, which could be potential targets for improving neurite outgrowth after injury.