Recently, we reported that a calix [6] arene carboxylic acid derivative can selectively extract the lysine-rich protein cytochrome c by interacting with amino groups on the protein surface. In the present article, quantitative extraction and recovery of cytochrome c using this calix[6]arene carboxylic acid derivative are described. Both adjustment of the pH under acidic conditions and addition of an alcohol are necessary to strip the extracted protein from an organic solution to an aqueous solution. Separation of cytochrome c and lysozyme using the calix[6]arene was achieved under the optimal conditions. In the forward extraction stage, 93% of the cytochrome c was extracted, while lysozyme remained in the solution. In the subsequent stripping stage, the extracted cytochrome c was quantitatively recovered in an aqueous solution. Finally, separation of these proteins, which have similar molecular weights and isoelectric points, was accomplished.