Phosvitin phosphopeptides (PPP) with high calcium binding capacity were prepared by a combination of high-temperature and low-pressure treatment， trypsin and thermophilic protease complex digestion. The optimum conditions for the preparation of PPP-Ca were determined to be pH 9.5 and a peptide-to-calcium mass ratio of 7:1. 97% calcium chelation was achieved under these conditions and PPP-Ca showed excellent stability in simulated gastrointestinal digestion. The monolayer model of Caco-2 cells was used to verify the calcium transport of PPP-Ca in vitro. The Caco-2 cell model was validated by cell morphology， electrical resistance and sodium fluorescein permeation， and MTT results showed that PPP-Ca exhibited low toxic effects on Caco-2 cells. The effects of PPP-Ca and PPP on the mass concentration of calcium ions transported and the activity of alkaline phosphatase (AKP) in Caco-2 cells were investigated， and the relative expression of CalbindinD9K m RNA was determined by RT-PCR. The results showed that PPP and PPP-Ca significantly increased AKP activity， increased calcium transport from (3.06±0.08) μg to (5.66±0.62) μg， and increased CalbindinD9K m RNA expression by 2-fold， thus enhancing calcium transport and absorption in the human intestine. The findings provide scientific evidence for the development of new calcium supplements and phosvitin phosphopeptides.