p14(ARF) is one of the major tumor suppressors conventionally identified both as the mdm2-binding molecule restoring p53 function in the nucleus, and as a nucleophosmin-binding partner inside the nucleolous to stabilize ribosomal RNA. However, its recently reported mitochondrial localization has pointed to novel properties as a tumor suppressor. At the same time, functional peptides are gaining much attention in nanomedicine for their in vivo utility as non-invasive biologics. We previously reported the p14(ARF)-specific peptide that restored the sensitivity to gefitinib on the gefitinib-resistant lung cancer cells. Based on the information of this prototype peptide, here we generated the more powerful anti-tumor peptide "r9-CatB-p14 MIS," which comprises the minimal inhibitory sequence of the mitochondrial targeting p14(ARF) protein in combination with the proteolytic cleavage site for cathepsin B, which is activated in various tumor cells, fused with the nine-polyarginine-domain for cell penetration, and demonstrated its novel action of regulating mitochondrial function in accordance with localization of endogenous p14(ARF). The p14 MIS peptide showed a potent tumor inhibiton in vitro and in vivo against not only lung cancer cells but also tumor cells of diverse lineages, via modulating mitochondrial membrane potential, with minimal cytotoxicity to non-neoplastic cells and tissues. Hence, this mitochondrially targeted p14 peptide agent provides a novel basis for non-invasive peptide-based antitumor therapeutics.