Multiple freeze-thaw (F-T) treatments could modify a protein structure and affect its physicochemical and biological activities. In this work, egg phosvitin (PSV) was subjected to multiple F-T treatments, and the changes in physicochemical and functional properties were investigated. The F-T treatments modified the molecular characteristics of PSV involving a decrease in surface hydrophobicity. Differential scanning calorimetry and scanning electron microscopy showed that PSV underwent denaturation, dissociation and possibly aggregation. Correspondingly, the emulsifying ability of PSV dramatically improved from 1.87 m2 g-1 to 3.70 m2 g-1, 3.25 m2 g-1 and 3.15 m2 g-1 after 3, 6, and 9 F-T cycles, respectively. In parallel, the PSV phosphopeptides (PPP) derived from the F-T treated PSV showed a higher calcium binding capacity and protecting activity against H2O2-induced apoptotic cell death of HepG2 cells, when compared with PPP from native PSV. These results indicated that the F-T treatments have potential to be implemented as a strategy to improve the emulsifying and biological activities of PSV.