Von Willebrand factor (VWF) is a large, multimeric plasma glycoprotein that critically mediates hemostasis at sites of vascular injury. Very large VWF multimers have the greatest thrombogenic activity, which is attenuated by cleavage in the A2 domain by the metalloproteinase ADAMTS13. ADAMTS13 proteolysis requires mechanical force to expose the scissile bond and is regulated by a calcium-binding site within A2. In this study, we characterized the interaction between VWF A2 and calcium by examining the effect of calcium on VWF A2 stability and mechanical unfolding and refolding. Isothermal calorimetry yielded a calcium binding K(d) = 3.8 ± 1.0 ?M and reversible thermal denaturation showed that 5 mM calcium stabilized the unfolding transition from 56.7 ± 0.1 to 69.1 ± 0.1 °C. Using optical tweezers to apply tensile force to single domains, we found that calcium did not affect VWF A2 unfolding, but rather enhanced refolding kinetics fivefold, resulting in a 0.9 kcal/mol stabilization in the folding activation energy in the presence of calcium. Taken together, our data demonstrate that VWF binds calcium at physiologic calcium concentrations and that calcium stabilizes VWF A2 by accelerating refolding.