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In this study, the antibacterial characteristics and mechanism of Cordyceps cicadae polysaccharide were determined. A water-soluble polysaccharide from Cordyceps cicadae was separated, and antibacterial activity against common pathogens was investigated by Oxford cup method. To investigate its antibacterial mechanism, the change of bacterial (Escherichia coli as the indicator bacteria) growth curve, electric conductivity, alkaline phosphatase (AKP) and β-galactosidase activity were measured. And the mycoprotein, membrane protein were also studied. The results showed that Cordyceps cicadae polysaccharide had strong antibacterial activity against Escherichia coli, Staphyloccocus aureus, Bacillus subtilis, Salmonella paratyphi and Pseudomonas aeruginosa. The minimum inhibitory concentration (MIC) to Escherichia coli was 0.10 mg/mL. In addition, the electric conductivity, AKP and β-galactosidase activity in microbial broth increased. The growth curve, mycoprotein, membrane protein changed. Our results revealed that Cordyceps cicadae polysaccharide exerted its bactericidal activity by damaging bacterial cell wall and cell membranes, increasing the cell permeability which resulted in the structural lesions and release of cell components, thus led to cell death.