Human centrin 2 (HsCen2) is an important calcium binding protein, which belongs to the conserved EF-hand superfamily. Reversible phosphorylation of protein is a crucial and ubiquitous regulatory mechanism in vivo. In the present paper, we investigated the binding of HsCen2 with the peptides of Kar1p, Rad4 and Sfi1 by isothermal titration calorimetry (ITC), fluorescence spectroscopy and native-PAGE. Based on fluorescence titration and ITC, the conditional dissociation constants of HsCen2 with the peptides were calculated. It is worth noting that HsCen2 displayed different binding modes with these three peptides. It reacted with Kar1p through calcium ions and phosphorylation double-regulation. And phosphorylation modification directed amino acid Ser170 played main role during HsCen2 binding with Rad4. As for peptide Sfi1, calcium ions or phosphorylation modifying Ser170 had little effects on their interaction. The native-PAGE indicated that a new band appeared corresponding to a complex of peptides binding to HsCen2. The binding of HsCen2 to calcium ions and especially phosphorylation of HsCen2 may change the hydrophobic cavities on HsCen2 and result in the changes of interaction of peptides with protein. These results are useful for us to understand the mechanism of phosphorylation regulation function in vivo.