Abstract: To develop a cell-penetrating peptide with high membrane penetrating ability and effective antitumor activity, we designed and synthesized an analogue of penetratin, Cys-CPT^2,9 penetratin, by substitution of Gln² and Asn⁹ with Cys-CPT. We investigated the transmembrane activity and antitumor activity of Cys-CPT^2,9 penetratin. The fluorescence intensity of Cys-CPT^2,9 penetratin in HeLa cells was observed by laser confocal microscopy and flow cytometry, and the cell uptake mechanism of Cys-CPT^2,9 penetratin was evaluated by using different endocytic inhibitors, finally the anti-tumor activity of Cys-CPT^2,9 penetratin was tested by MTT assay. The results showed that the membrane activity of Cys-CPT^2,9 penetratin was significantly enhanced in laser confocal microscopy and flow cytometry assay, and the intracellular fluorescence intensity was 5 times higher than penetratin. The cell uptake mechanism study of Cys-CPT^2,9 penetratin indicated that it mainly entered the cell through the clathrin and endocytosis. Moreover, Cys-CPT^2,9 penetratin exhibited anti-tumor activity against HeLa cells, and its inhibitory effect on cancer cells was stronger than CPT. Cys-CPT^2,9 penetratin is a new cell-penetrating peptide with high translocation ability, and has anti-tumor activity against HeLa cells.