Lysozyme was encapsulated within biodegradable poly(D,L-lactide-co-glycolide) microspheres by a double emulsion solvent evaporation method for studying its release mechanism associated with protein stability problems. When urea, a protein unfolding agent, was added into the incubation medium lysozyme release rate from the microspheres increased with the increase in urea concentration. The enhanced lysozyme release was attributed to the suppression of protein aggregation, to the facilitated diffusion of unfolded lysozyme by an efficient reptile motion of unfolded protein molecules through porous channels in microspheres, and to the largely decreased extent of nonspecific protein adsorption onto the enlarged surface area of degrading polymer microspheres in the presence of urea. Encapsulating lysozyme in an unfolded form within PLGA microspheres was attempted by using urea as an excipient. This new urea-based formulation exhibited a more sustained lysozyme release profile than the control formulation, and released lysozyme from the microspheres showed a much less amount of lysozyme dimer population while maintaining a correct conformation after refolding in the incubation medium. This study provides new insights for the formulation of protein encapsulated PLGA microspheres.