Calcium- and Integrin-Binding protein 2 (CIB2) is a widely expressed protein with an uncertain biological role. Two of its four EF-hand motifs bind Mg(II) and/or Ca(II), thus triggering conformational changes. Although previous studies suggested that CIB2 preferentially binds Mg(II) over Ca(II) under physiological conditions, an atomic level characterization of CIB2 in the presence of both cations was lacking. Based on a combination of solution NMR, exhaustive molecular dynamics simulations and isothermal titration and differential scanning calorimetry, we characterized the interaction of CIB2 with both Ca(II) and Mg(II) ions and elucidated the protein regions involved in the interaction with the α7B integrin target. Analysis of experimental amide nitrogen relaxation rates shows that the EF4 motif exhibits high mobility regardless of the specific bound metal ion and demonstrates that the Mg(II)- and Ca(II)-bound state of CIB2 is relatively floppy, with pico-nanosecond motions induced in a region involved in target recognition. Overall, our data indicate a preferential, thermodynamically stable but structurally flexible state for CIB2, in which a Mg(II) ion is bound to EF3 and a Ca(II) ion to EF4. These results unveil the role of metal binding events in CIB2 and offer new insights into the dynamic regulation of target recognition.