The avidity of cancer cells for iron highlights the potential for iron chelators to be used in cancer therapy. Herein, we designed and synthesized a novel series of 5H-[1,2,4]triazino[5,6-b]indole derivatives bearing a pyridinocycloalkyl moiety using a ring-fusion strategy based on the structure of an iron chelator, VLX600. The antiproliferative activity evaluation against cancer cells and normal cells led to the identification of compound 3k, which displayed the strongest antiproliferative activity in vitro against A549, MCF-7, Hela and HepG-2 with IC50 values of 0.59, 0.86, 1.31 and 0.92 μM, respectively, and had lower cytotoxicity against HEK293 than VLX600. Further investigations revealed that unlike VLX600, compound 3k selectively bound to ferrous ions, but not to ferric ions, and addition of Fe2+ abolished the cytotoxicity of 3k. Flow cytometry assays demonstrated that 3k arrested the cell cycle at the G1 phase and induced significant apoptosis in A549 cells in dose and time-dependent manners, corresponding to JC-1 staining assay results. Western blot analysis of Bcl-2, Bax and cleaved caspase-3 proteins further provided evidences that induction of apoptosis by 3k in A549 cells might be at least via the mitochondria pathway. These above results highlight that 3k is a valuable lead compound that deserves further investigation as an iron chelator for the treatment of cancer.