Tetrabutylphosphonium N-trifluoromethanesulfonyl leucine ([P₄₄₄₄][Tf-Leu], IL) shows phase separation with water at 25 °C, but is miscible at 20 °C. Such a reversible phase transition, called lower critical solution temperature (LCST) behaviour, has been utilised to extract proteins. An aqueous solution of cytochrome c (Cyt. c) was mixed with an equal volume of the IL and the solution was cooled down to 20 °C to obtain a homogeneous solution. After phase separation was induced by heating it at 25 °C, all the Cyt. c was found in the IL phase. The IL, containing at least 21 wt% of water, was effective to extract Cyt. c. The distribution ratio (D) of several proteins in the IL/water mixture depended strongly on the isoelectric point (pI) of the corresponding proteins. Based on the difference in the D value, separation of a target protein from their mixtures was successfully carried out using the LCST behaviour of the IL/water mixture.