From a series of solubilization experiments for various surfactants with two alkyl chains, di(2-ethylhexyl)phosphorothioic acid (DEPTA) is found to be effective for the formation of reverse micelles (RMs). The DEPTA/isooctane/water system can extract and concentrate hemoglobin as a comparatively large molecular weight protein. The size and shape of the RM were determined by small angle X-ray scattering (SAXS) method; it has been demonstrated that DEPTA assumes a spherical shape just like the conventional Aerosol-OT. Furthermore, desolubilization of the protein entrapped in the RMs by weak alkaline solution can be realized with the addition of a small amount of n-octanol, and then the micellar solution after stripping can be regenerated by bringing it into contact with dilute hydrochloric acid. It is illustrated in the recovery of hemoglobin that the present RM system can possibly be used in the extraction process in cascade operation.