Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca 2+ binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With D63N S100B as an exception ( D63N K D =50±9μM), Ca 2+ binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 ( D61N K D =412±67μM, D65N K D =968±171μM, and E72A K D =471±133μM), when compared to wild-type protein ( WT K D =56±9μM). For the TRTK-12 complexes, the Ca 2+ -binding affinity to wild type ( WT+TRTK K D =12±10μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target ( D61N+TRTK K D =29±1.2μM, D63N+TRTK K D =10±2.2μM, D65N+TRTK K D =73±4.4μM, and E72A+TRTK K D =18±3.7μM). In addition, R ex , as measured using relaxation dispersion for side‐chain 15 N resonances of Asn63 ( D63N S100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, D61N S100B, D63N S100B, and D65N S100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca 2+ -bound (2.0Å) and TRTK-bound (1.2Å) D63N S100B showed no change in Ca 2+ coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca 2+ -binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca 2+ binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.