Objective: To research the activity of antibacterial proteins isolated from Musca domestica during larvae-pupae metamorphosis. Methods: The 5-day-old larvae of Musca domestica were injured with a needle. 24h later, the larvae which had changed into pupae were selected. The antibacterial protein was isolated by a four-step protocol including grinding, heating, CM-sepharose F. F. cationexchange chromatography and another CM-sepharose F. F. chromatography. Antibacterial activities of samples were tested by an ultrasensitive radial diffusion assay using Staphylococcus aureus as the indicator organism. The molecular weight of the isolated protein was determined by SDS-PAGE.Results: The molecular weight of this isolated protein was 43kDa, and its antibacterial activity was strong. Conclusions: A kind of protein with strong antibacterial activity can be produced in the Musca domestica during larvae-pupae metamorphosis.