When a protein is encapsulated into poly( DL -lactide-co-glycolide)(PLGA) microspheres by means of the double-emulsion method,the harsh microspheres formation process including ultrasonification,exposure to an organic solvent and a polymer may cause the denaturation of the protein. In this study,we investigated the enzymatic activity change and the effect of the excipients on the stability of recombinant human Cu,Zn-superoxide dismutase(rhCu,Zn-SOD) during the emulsification. The specific activity recovery was found to be concentration dependent and the excipients involved such as PEG 600 and Tween 20,and trehalose were shown to increase the stability of rhCu,Zn-SOD. The protein structural integrity within the microspheres was analyzed by FTIR. The structure of rhCu,Zn-SOD within PLGA microspheres containing trehalose was found to be similar to that of the native solid state,whereas the protein encapsulated during the preparation in the absence of any excipient changed due to the possible hydrophobic interaction with the polymer. The results suggest that a rational stability strategy for protein to be encapsulated into microspheres should aim at different processes. When a protein is encapsulated into poly (DL -lactide-co-glycolide) (PLGA) microspheres by means of the double-emulsion method, the harsh microspheres formation process including ultrasonification, exposure to an organic solvent and a polymer may cause the denaturation of the protein. In this study, we investigated the enzymatic activity change and the effect of the excipients on the stability of recombinant human Cu, Zn-superoxide dismutase (rhCu, Zn-SOD) during the emulsification. The specific activity was was found to be concentration dependent and the excipients involved such as PEG 600 and Tween 20, and trehalose were shown to increase the stability of rhCu, Zn-SOD. The protein structural integrity within the microspheres was analyzed by FTIR. The structure of rhCu, Zn-SOD within PLGA microspheres containing trehalose was found to be similar to that of the native solid state, whereas the protein encapsulated during the preparation of the absence of any excipient changed due to the possi ble hydrophobic interaction with the polymer. The results suggest that a rational stability strategy for protein to be encapsulated into microspheres should aim at different processes.