The effects of salt concentration in mobile phase, elution strategy, and hydrophobicity of stationary phase on lysozyme refolding in hydrophobic interaction chromatography (HIC) were investigated. Butyl Sepharose 4 Fast Flow, the least hydrophobic HIC resin among the tested adsorbent, showed the best refolding yield. The binding efficiency of unfolded lysozyme on the adsorbent was maximized when 1 and 0.4 M of initial and final concentration of ammonium sulfate was used in mobile phase, respectively. The optimum gradient strategy for refolding and elution of lysozyme was determined as linear increase of urea concentration to 4M. The optimized condition suggests the less hydrophobic environment than conventionally used salt solutions and HIC resins. Consequently, total refolding yield was improved 1.6 times comparing with optimized dilution-based batch refolding method.