Abstract: This study aimed to explore the physiological activity of phosphorylated soybean polypeptide chelated calcium (PSPCC) and improve its calcium chelation capacity. To be specific, the preparation process of PSPCC was optimized by single factor test and response surface methodology. And the effect of phosphorylated soybean peptide chelated calcium on osteoblast activity was evaluated by in vitro experiments. The results showed that, the optimum process of PSPCC was as follows: Mass ratio of sodium tripolyphosphate to soybean polypeptide 1:2, phosphorylation reaction temperature 52 ℃, phosphorylation reaction pH7.0, phosphorylation reaction time 9.7 h, mass ratio of phosphorylated soybean polypeptide to calcium chloride 2:1, chelation reaction pH8.0, chelation reaction temperature 50 °C, and chelation reaction time 1.5 h. The maximum calcium chelation amount of PSPCC was 107.25±0.10 mg/g under the above optimal conditions. The results of MTT assay showed that the relative proliferation rate of osteoblasts in the PSPCC group (group D) was 1.6 times that of the soybean polypeptide group (group A) (the third day). In particular, the ALP staining experiment showed that the positive rate of staining in group D was 33.6 times that of the blank group. In addition, the ALP activity of each sample was detected by the ALP kit on the 7th day, and the results showed that the ALP activity of the group D was 6.2 times that of the blank group. And it was found that the number of calcium nodules in the group D was 94 times higher than that of the blank group through the alizarin red staining experiment. It was reasonable that the preparation process of PSPCC was optimized by response surface methodology, which was preliminarily proved that PSPCC had a significant effect on promoting proliferation and differentiation of osteoblasts (P<0.05), and the effect of PSPCC on promoting proliferation and differentiation was higher than that of other samples. This result could provide a theoretical basis for the further exploitation and utilization of PSPCC.