10-HDA(10-hydroxy-2-decenoic acid), an unsaturatedmedium-chain fatty acid in royal jelly, has been shown to possess biological activity in many fields, including the prevention of cardiovascular disease, cancer, bacteria and radiation. Inthis study, the antibacterial activity of 10-HDA againstE.coli was evaluated. Meanwhile, the acting mechanism of 10- HDA on E. coli was investigated by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with 10-HDA. The experimental results indicated 0.25 mg/ml 10-HDA could completely inhibit the growth of E.coliwhich dissolves in liquid Luria-Bertani (LB) medium. Meanwhile, 10-HDA resulted in the leakage of alkaline phosphatase, changedionic conductivity, and induced the respiratory chain dehydrogenases into inactive state, When E. coliwere exposed to 1.0 mg/ml 10-HDA, the surface of the cells were roughand shrank obviouslyobserved by atomic force microscope (AFM), indicating the bacterial membranous structure was damaged severely. In conclusion, the combined results suggested that 10-HDA may damage the structure of bacterial cellmembrane, which cause E.coli bacteria to die eventually.