The S100A1 protein mediates a wide variety of physiological processes through its binding of calcium (Ca2+) and endogenous target proteins. S100A1 presents two Ca2+-binding domains: a high-affinity “canonical” EF (cEF) hand and a low-affinity “pseudo” EF (pEF) hand. Accumulating evidence suggests that both Ca2+-binding sites must be saturated to stabilize an open state conducive to peptide recognition, yet the pEF hand’s low affinity limits Ca2+ binding at normal physiological concentrations. To understand the molecular basis of Ca2+ binding and open-state stabilization, we performed 100 ns molecular dynamics simulations of S100A1 in the apo/holo (Ca2+-free/bound) states and a half-saturated state, for which only the cEF sites are Ca2+-bound. Our simulations indicate that the pattern of oxygen coordination about Ca2+ in the cEF relative to the pEF site contributes to the former’s higher affinity, whereas Ca2+ binding strongly reshapes the protein’s conformational dynamics by disrupting β-sheet coupling between EF hands. Moreover, modeling of the half-saturated configuration suggests that the open state is unstable and reverts toward a closed state in the absence of the pEF Ca2+ ion. These findings indicate that Ca2+ binding at the cEF site alone is insufficient to stabilize opening; thus, posttranslational modification of the protein may be required for target peptide binding at subsaturating intracellular Ca2+ levels.