Several diacerein α-aminophosphonates conjugates 4a-k have been synthesized, the structures of compounds have been characterized by IR, H NMR, C NMR, P NMR, ESI-MS spectra and elementary analysis. In vitro cytotoxicity against HepG-2, CNE, Spca-2 and Hct-116 cells are evaluated and employing standard MTT assay in comparing with commercial anticancer drug 5-fluorouracil (5-FU). Some compounds exhibit moderate to high levels of antitumor activity. Especially, compound 4i exhibit the strongest cytotoxicity against Hct-116 cells with IC50 9.83 μM. All the synthesized compounds exhibit low cytotoxicity against HUVEC cells. The mechanism of compound 4i has been preliminarily investigated by Hoechst 33258 staining, JC-1 mitochondrial membrane potential staining and flow cytometry, which indicate that the compound 4i induced apoptosis in Hct-116 cancer cells. Cell cycle analysis show that compound 4i mainly arrested Hct-116 cells in G1 stage. The effects of 4i on the activation of caspases expression indicate that 4i might induce apoptosis via the membrane death receptor pathways. In addition, the binding properties of a model analog 4i to DNA have been investigated by methods (UV-Vis, fluorescence, CD spectroscopy) in comparison with that of diacerein. Results indicate that 4i show moderate ability to interact ct-DNA.