Abstract: Objective To evaluate the anticancer activity of newly synthesized phenazine derivatives and investigate the possible mechanism. Methods Cell proliferation was tested by MTT assay. The morphology of HCT116 cells was observed by fluorescent microscope after acridine orange/ethidium bromide(AO/EB) dye. Cell cycle distribution was detected by flow cytometry, and cell apoptosis was examined by Annexin V-FITC/7-AAD staining. The expressions of p53 and caspase-3 proteins were detected by Western blot. Results All compounds inhibited the growth of cancer cells, especially human colorectal cancer cells HCT116, in a time- and dose-dependent manner. Compound pc28 showed the most potent antiproliferation effect on HepG2, HCT116 and A549 cells with half maximal inhibitory concentration(IC50) being (6.62±2.69), (10.83±1.41) and (22.39±4.31)μmol respectively at 48h. 20μmol compound on HCT116 cells for 24h resulted in the decrease of cell density, morphological change to round, increase of green fluorescence related to chromatin condensation, and dead cells showed orange or red color implying cell membrane permeability increase. Besides, pc28 induced apoptosis rate of HCT116 cells in advanced stage from 0.345% to 19.7%. 20μmol pn18 and pc27 induced the increase of cell number in G0/G1 phase for 17.5% and 25.0% respectively, while pn23 and pc28 induced the increase of cell number in S phase for 18.4% and 11.0%. Compound pn23 and pc28 elevated p53 protein expression. pc28 also upregulated and activated caspase-3. Conclusion The newly synthesized phenazine derivatives show effective anticancer activity in vitro, and the mechanism might be related to inducing cell cycle arrest and apoptosis.