It is believed that denatured-reduced lysozyme rapidly forms aggregates during refolding process, which is often worked around by operating at low protein concentrations or in the presence of aggregation inhibitors. However, we found that low concentration buffer alone could efficiently suppress aggregation. Based on this finding, stable equilibrium intermediate states of denatured-reduced lysozyme containing eight free SH groups were obtained in the absence of redox reagents in buffer of low concentrations alone at neutral or mildly alkaline pH. Transition in the secondary structure of the intermediate from native-like to beta-sheet was observed by circular dichroism (CD) as conditions were varied. Dynamic light scattering and ANS-binding studies showed that the self-association accompanied the conformational change and the structure rich in beta-sheet was the intermediate state for aggregation, which could form either amyloid protofibril or amorphous aggregates under different conditions as detected by Electron Microscopy. Combining the results obtained from activity analysis, RP-HPLC and CD, we show that the activity recovery was closely related to the conformation of the refolding intermediate, and buffer of very low concentration (e.g. 10mM) alone could efficiently promote correct refolding by maintaining the native-like secondary structure of the intermediate state. This study reveals reasons for lysozyme aggregation and puts new insights into protein and inclusion body refolding.