The structure of thrombin-binding DNA aptamer complexed with a single Sr²⁺ ion (Sr²⁺:TBA complex) has been determined using NMR spectroscopy and restrained molecular dynamics simulations. The quadruplex structure for the Sr²⁺:TBA complex is similar in topology, but distinct in structure, from that previously reported for the K⁺:TBA complex. The inter-tetrad distance of the Sr²⁺:TBA complex is 3.8 Å, or 0.7 Å larger than in the K⁺:TBA complex. This substantial difference can be attributed to a different binding site for Sr²⁺ in the Sr²⁺:TBA complex than for K⁺ in the K⁺:TBA complex. The Sr²⁺:TBA complex assumes a 1:1 stoichiometry, and it is very likely that the Sr²⁺ ion simultaneously interacts with the eight O6 atoms of the two G-tetrads. The results indicate that quadruplex DNA structures are highly sensitive to the presence of specific metal ions. The binding of specific metal ions may modulate the biological activity of quadruplex DNA structures in vivo.