Calgranulin C performs antimicrobial activity in the human immune response by sequestering Zn(II). This biological function is afforded with the aid of two structurally distinct Ca(II)‐binding EF hand motifs, wherein one of which bears an unusual amino acid sequence. Here, we utilize solution state NMR relaxation measurements to investigate the mechanism of Ca(II)‐modulated enhancement of Zn(II) sequestration by calgranulin C. Using C13/N15 CPMG dispersion experiments we have measured pH‐dependent major and minor state populations exchanging on micro‐to‐millisecond timescale. This conformational exchange takes place exclusively in the Ca(II)‐bound state and can be mapped to residues located in the EF‐I loop and the linker between the tandem EF hands. Molecular dynamics (MD) simulations spanning nano‐to‐microsecond timescale offer insights into the role of pH‐dependent electrostatic interactions in EF‐hand dynamics. Our results suggest a pH‐regulated dynamic equilibrium of conformations that explore a range of “closed” and partially “open” sidechain configurations within the Zn(II) binding site. We propose a novel mechanism by which Ca(II) binding to a non‐canonical EF loop regulates its flexibility and tunes the antimicrobial activity of calgranulin C.