The in vitro insulin unfolding had been studied using the “equilibrium unfolding” method where protein is unfolded by reducing reagents in the presence of trace amounts of oxidants such as oxidized glutathione. Nine intermediates were captured in the unfolding process, named as P1A, P2A, P3A, P4A, P3B, P4B, P5B, P6B, and P7B, which were all either A chain derivatives or B chain derivatives. No intermediate with inter-A-B chain disulfide was captured. Based on the character of the intermediates, their distribution during the unfolding process and the hypothetic “transient” intermediates, an in vitro putative unfolding pathway of insulin had been proposed. Besides, the comparison of the intermediates captured in unfolding with the intermediates captured in the refolding process of insulin revealed that both unfolding/ refolding processes of insulin shared common intermediates. Based on these observations we suggested that the unfolding pathway of insulin was similar to the refolding pathway but flowed in the opposite direction.