Abstract: Investigations on protein refolding by size-exclusion chromatography (SEC) was carried out by using lysozyme as a model system.The comparison of elution volume of the native lysozyme and the refolded lysozyme demonstrated the variation of protein diameters during its refolding.The high yield of lysozyme activity obtained at high starting concentration illustrates the workability of this method for high concentration protein refolding.The effects of the sample loading and elution flowrate on the recovery of lysozyme were investigated,separately.The circular dichroism analysis demonstrated the structural identity of the refolded lysozyme and the native lysozyme.Short columns were used for the investigation of the refolding kinetic,and the impact of separation on refolding.The experimental results described above showed the advantages of this new method over the conventional step-wise dilution procedure in terms of high sample concentration and the in situ polishing and refolding.