The interleukin-1 receptor antagonist (IL-1Ra), having the potential of treating cancers, has been used for treating rheumatoid arthritis in clinic. To improve its therapeutic efficacy and patient compliance, IL-1Ra was often needed to prepare a long efficacy formulation using all kinds of method for protein stabilization. In this study, we reported a novel freezing-induced PEG water solution phase/dextran water solution phase separation method to prepare the IL-1Ra-loaded dextran microparticles to address stability of IL-1Ra in preparation process for all kinds of formulation, namely: first IL-1Ra was dissolved in water together with dextran and polyethylene glycol, and then by a freezing process during which dextran is separated out as the dispersed phase with IL-1Ra partitioned in dextran microparticles preferentially. The frozen sample was then lyophilized to powder and washed with dichloromethane to remove the PEG continuous phase. Once IL-Ra was loaded in the dextran microparticles, 1-5 mu m in diameter, the dextran microparticles gained resistance to oil/water interfaces without IL-Ra aggregation and activity loss. The bioactivity from dextran microparticles in vitro is preserved highly (above 98%), while bioactivity from IL-1Ra-loaded dextran micropartides using the control method of water-in-oil-in-water (W/O/W) method is only 70.5% by inhibiting melanoma B16 cell proliferation. The dextran microparticles may be used further to prepare different formulations according to clinic needs. 2012 Elsevier B.V. All rights reserved.