Cell-penetrating peptides (CPPs) have a great potential for intracellular delivery of cell-impermeable biological macromolecules in clinical therapy. However, their lack of cell and tissue specificity remains the primary limitation for their clinical development as drug delivery vehicles. In this study, based on phage display and an in silica approach, we found a novel CPP-MT23 with mouse melanoma cell specificity, it can only enter B16 melanoma cancer cells and without any cytotoxicity, Moreover, MT23 showed higher penetration efficiency based on fluorescence microcopy and quantitative assay, and it has capability for mediating functional Apoptin into cells in vitro or in vivo. Moreover, MT23-Apoptin can significantly inhibit tumor growth and induce the cell apoptosis in B16 tumor bearing mice. To sum up, all the results implicated that MT23 has the potential to deliver exogenous therapeutic proteins for further use and it also expected to lay the foundation for developing human melanoma cancer cell specific CPP.