The purpose of the present paper is to study Ca2+ transmembrane behaviors of the S.aureus treated with high intensity pulsed magnetic field. For this purpose, the method of Fura-2/AM fluorescence probe was investigated to determine the change in intracellular Ca2+ concentration in S.aureus, fluorescence intensities of S.aureus cells treated by pulsed magnetic field under different pulse number were determined, and the change in intracellular Ca2+ concentration was observed by laser confocal scanning microscope (LCSM). Research results showed that Fura-2/AM can load in S.aureus successfuuly, and can be used to determine the change in intracellular free Ca2+ concentration. After being treated with high intensity pulsed magnetic field, intracellular free Ca2+ concentration of S.aureus went up significantly, and the regulation of Ca2+ increase was close consistent with the decrease in living bacterium number, and relatedness reached to -0.989 15; the number and fluorescence intensity of intracellular lightspot increased significantly, meaning that large amount of exocellular calcium ions across cell membrane enter into inside of cells. So, it was judged that the change in microbial membrane permeability and increase in intracellular free Ca2+ concentration are important reasones why high intensity pulsed magnetic field has sterilization effect.