Practical methods to deliver proteins systemically in animals have been hampered by poor tissue penetration and inefficient cytoplasmic localization of internalized proteins. We therefore pursued the development of improved macromolecule transduction domains (MTDs) and tested their ability to deliver therapeutically active p18(INK4c). MTD103 was identified from a screen of 1,500 signal peptides; tested for the ability to promote protein uptake by cells and tissues; and analyzed with regard to the mechanism of protein uptake and the delivery of biologically active p18(INK4c) into cancer cells. The therapeutic potential of cell-permeable MTD103p18(INK4c) (CP-p18(INK4c)) was tested in the HCT116 tumor xenograft model. MTD103p18(INK4c) appeared to traverse plasma membranes directly, was transferred from cell-to-cell and was therapeutically effective against cancer xenografts, inhibiting tumor growth by 86-98% after 5 weeks (P < 0.05). The therapeutic responses to CP-p18(INK4c) were accompanied by high levels of apoptosis in tumor cells. In addition to enhancing systemic delivery of CP-p18(INK4c) to normal tissues and cancer xenografts, the MTD103 sequence delayed protein clearance from the blood, liver and spleen. These results demonstrate that macromolecule intracellular transduction technology (MITT), enabled by MTDs, may provide novel protein therapies against cancer and other diseases.