Cell membranes are a great obstacle for entrance of gene therapeutic agents. Cell-penetrating peptides (CPPs) have been proven as a promising gene delivery tool. However, the early TAT peptide derived from the HIV transcription activator protein has been proven that the sequence contains Furin protease cleaved motifs which limited the TAT application in delivery of exogenous active molecules. In the present study, through the bioinformatics and experimental approach, we have identified a novel CPP derived from the N terminus of VP1 protein of chicken anemia virus (CAV), designated as CVP1-N2, which is rich in arginine residues and contains α-helical structure. Then, the ability of CVP1-N2 cell penetrating was detected using confocal imaging and flow cytometry. FITC-labeled CVP1-N2 peptide could rapidly internalize into different types of live cells with dose dependence and without cytotoxic effects by MTT assay. Surprisingly, CVP1-N2 with a pattern of nuclear sub-location has shown the higher uptake efficiency than TAT. At 10, 1, and 0.1 μM, the mean relative internalization of CVP1-N2 was respectively 1.08-, 12-, and 75-fold higher than that of CVP1, as well as 1.6-, 56-, and 75-fold higher than that of TAT. Moreover, using endocytic inhibitors along with low-temperature stress validated that the CVP1-N2 internalization route is direct translocation pathway. Finally, the capacity of CVP1-N2 for delivery of gene into cells was determined, where it was able to carry red fluorescent protein (RFP) and apoptin genes into cells respectively and induce the apoptosis. All these data indicate that CVP1-N2 could be used as a novel gene delivery vehicle for gene therapy in the future. KEY POINTS: • 1CVP1-N2 was identified as a novel more efficient cell-penetrating peptide. • 2. CVP1-N2 localized to the nucleus through the direct transduction pathway. • 3. CVP1-N2 was able to deliver the apoptin gene into HCT116 cells and induce apoptosis.