Two isomeric Zinc (II) complexes constructed by 3,5-bis(1-imidazoly) pyridine has been synthesized and characterized by single crystal X-ray diffraction, elemental analyses and infrared spectroscopy. The binding mode and ability of complex 1-2 with CT-DNA were studied by UV and fluorescence spectra. The intrinsic binding constant K b (K b1 = 2.305 × 10 4 M −1 , K b2 = 3.095 × 10 4 M −1 ) and the observed association constant K obs (K obs1 = 1.523*10 6 M −1 , K obs2 = 2.057*10 6 M −1 ) indicated that the insertion ability of complex 2 with CT-DNA is stronger than complex 1. Gel electrophoresis showed that complexes have a good ability to hydrolyze cleavage pBR322 plasmid DNA. The cytotoxicity and apoptosis studies showed that complexes exhibited excellent cytotoxic activity against HeLa cells, especially complex 2 had better growth inhibition than Cisplatin. Molecular docking study simulated the binding model of complexes with DNA (PDB:4av1), showing an imidazole plane of complex 2 can be inserted into a DNA base pair in relative parallel. Both complexes can be used as potential anticancer agents.