Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. The first amino acid of the Ca2+-binding loops found in the EF-hand calcium-binding proteins is a highly conserved aspartic acid residue. The D37K mutant was produced to elucidate the metal binding role of the first aspartic acid of the EF-loop I of EoCen. Aromatic-sensitized Tb3+ fluorescence results indicated that the metal binding ability of loop I was lost due to the D37K mutation. Based on fluorescence titration curves of Lu2-D37K, the conditional binding constants of the EoCen loop II were quantitatively found to be KII = (1.61 ± 0.04) × 105 L mol−1 and KII = (3.52 ± 0.08) × 102 L mol−1 with Tb3+ and Ca2+, respectively. Using 2-p-toluidinylnaphthalene-6-sulfonate as a hydrophobic probe, exposure of the hydrophobic surface upon metal binding was found to be significantly reduced for the metal ion-saturated EoCen D37K mutant.