The retention behavior of nine proteins was experimentally detected by using high performance size exclusion chromatography (SEC). At low ionic strength of mobile phase, the retention behavior of proteins is dependent of not only size exclusion but also electrostatic interaction. At the same pH value of mobile phase as the isoelectric point of a protein, the protein molecule is neutral. When the pH value of mobile phase is higher than the isoelectric point of a protein, the protein molecule carries a negative charge, resulting in size exclusion and ionic exclusion occuring together. Otherwise, ionic adsorption dominates in electrostatic interaction. At middle ionic strength of mobile phase, the retention behavior is governed by size exclusion mechanism. The experimental result indicated that in order to achieve ideal size exclusion the elution conditions for proteins should be ionic strength 0.1∼0.3 M and pH value near its isoelectric point. A linear “calibration curve” was correlated by eight proteins except hemoglobin, in which the related coefficient was as high as 0.9977.