An improved method of refolding recombinant human proinsulin from E. coli was presented. It was based on a two-stage stirred tank reactor in which denatured proinsulin-s-sulfonate was mixed instantaneously with a reaction buffer in the first stage reactor, and then fed to the second stage reactor. The mixture was stirred further for a total of 30h in the second stage reactor. In this system, unfavorable effects present due to the increase in reaction volume and protein concentration for protein refolding, which becomes significant in a large-scale operation, were avoided. Refolding yields of over 80% was obtained for achieving reaction volume of upto 50 l at protein concentration of 1 mg/ml. The optimum urea concentration was 1M. Refolding yield at the 1-1 reaction volume and protein concentration of 0.5mg/ml was increased about 2.5-fold, compared to that in a batch reactor. By increasing protein concentration in a two-stage refolding reaction, the cost for insulin production could be reduced, therefore, making this process economical.