The inherent limitations, including serious side-effects and drug resistance, of current chemotherapies necessitate the search for alternative treatments especially for lung cancer. Herein, the anticancer activity of colicin N, bacteria-produced antibiotic peptide, was investigated in various human lung cancer cells. After 24 h of treatment, colicin N at 5–15 µM selectively caused cytotoxicity detected by MTT assay in human lung cancer H460, H292 and H23 cells with no noticeable cell death in human dermal papilla DPCs cells. Flow cytometry analysis of annexin V-FITC/propidium iodide indicated that colicin N primarily induced apoptosis in human lung cancer cells. The activation of extrinsic apoptosis evidenced with the reduction of c-FLIP and caspase-8, as well as the modulation of intrinsic apoptosis signaling proteins including Bax and Mcl-1 were observed via Western blot analysis in lung cancer cells cultured with colicin N (10–15 µM) for 12 h. Moreover, 5–15 µM of colicin N down-regulated the expression of activated Akt (p-Akt) and its upstream survival molecules, integrin β1 and αV in human lung cancer cells. Taken together, colicin N exhibits selective anticancer activity associated with suppression of integrin-modulated survival which potentiate the development of a novel therapy with high safety profile for treatment of human lung cancer.