In conclusion, this work contributed to a better understanding of the chelation chemistry of kaempferol with Zn(II) ion and the higher anticancer effect of the complex against tumor cells. The kaempferol-Zn was synthesized in a method of high-efficiency and the proposed chemical structure is characterized by spectroscopic analysis, which indicated that the chelation sites of kaempferol with Zn(II) ion were 3-OH and 4-oxo groups. Besides, kaempferol-Zn displayed two times higher anti-tumor activity against EC9706 cells than that of free kaempferol. The flow cytometric analysis provided clear evidence that the complex is able to increase intracellular calcium ion level in cancer cells. Furthermore, AFM morphological data showed that the complex could deform and shrink EC9706 cells at nanoscale. Collectively, all the data proved the as-synthesized kaempferol-Zn could be served as a promising pharmacological substance for anticancer treatment.