OBJECTIVES: p27Kip1 is a member of cyclin-dependent kinase (Cdk) inhibitors. Its overexpression results in cell cycle arrest in G1 and/or apoptosis. Taken together, inactivation of the cell cycle inhibitory activity of p27 is commonly observed in various cancers. We developed cell permeable p27 (CP-p27) fused to new-established novel Macromolecule Transduction Domain (MTD) can excessively transport macromolecule into living cells and animals. This research was to develop CP-p27 as an anti-cancer agent.  METHODS: The ability of penetrating into cells of CP-p27 was determined by flow cytometry and confocal laser scanning microscope. To evaluate whether CP-p27 regulate cell proliferation and cell cycle, the level of various proteins were analyzed by western blot analysis. Also the proliferation and viability of cancer cells were evaluated by crystal violet staining. RESULTS: Treatment of CP-p27 proteins in colon cancer cells (HCT 116) reduced phosphorylation of MEK and ERK and increased expression of cell-cycle inhibitor, p21. Furthermore, the cleavage of caspase 7 which was a marker of apoptosis was detected. In addition, viability of cancer cells was reduced significantly in presence of CP-p27. CONCLUSIONS: These results suggest that that CP-p27 could be protein-based biotherapeutic agent as an anti-cancer agent.