The back-extraction of proteins encapsulated in AOT reverse micelles was performed by adding a counterionic surfactant, either TOMAC or DTAB. This novel backward transfer method gave higher backward extraction yields compared to the conventional method with high salt and high pH of the aqueous stripping solution. The protein activity was maintained in the resulting aqueous phase, which in this case had a near neutral pH and low salt concentration. A sharp decrease of the water content was observed in the organic phase corresponding to protein back-extraction using TOMAC. The backward transfer mechanism was postulated to be caused by electrostatic interaction between oppositely charged surfactant molecules, which lead to the collapse of the reverse micelles. The back-extraction process with TOMAC was found to be very fast; more than 100 times faster than back-extraction with the conventional method, and as much as 3 times faster than forward extraction. The formation of 1:1 complexes of AOT and TOMAC in the solvent phase was observed, and these hydrophobic complexes could be efficiently removed from the solvent using adsorption onto Montmorillonite in order for the organic solvent to be reused. A second cationic surfactant, DTAB, confirmed the general applicability of counterionic surfactants for the backward transfer of proteins.