The Nogo-66 receptor (NgR1), a receptor for Nogo-A, contributes to the inhibition of axonal regeneration in the adult central nervous system after traumatic injuries. Thus, NgR1 has been considered a critical target in axon regeneration therapy. Here, we identified a specific NgR1 antagonist peptide (HIYTALV, named NAP2) which promotes neurite regeneration in vitro from a phage display heptapeptide library. NAP2 was co-localized with NgR1 on the surface of PC12 cells and cerebellar granule cells (CGCs) by immunofluorescence assay. Horseradish peroxidase (HRP)-streptavidin-biotin assay further showed that NAP2 binds to NgR1 and the dissociation constant (Kd) was 0.45. μM Functional analyses indicated that NAP2 could reduce the inhibitory effects of Nogo-66 on neurite outgrowth in differentiated PC12 cells and CGCs by blocking the Nogo-66-induced activation of Rho-associated coiled coil-containing protein kinase (ROCK), collapsin response mediator protein 2 (CRMP2) and myosin light chain (MLC). Taken together, the small molecule NgR1 antagonist peptide NAP2 (MW: 815.98. Da) has a potential ability in crossing blood brain barrier and will be a promising therapeutic agent for the treatment of spinal cord injury and neurodegenerative diseases.