With homology modeling techniques, molecular mechanics, and molecular dynamics methods, a 3D structure model of Ndx1 is created and refined. This model is further assessed by Profile-3D and ProStat, which confirm that the refined model is reliable. With this model, a flexible docking study is performed and the result indicates that Glu46, Arg88, and Glu90 are three important determinant residues in binding, as they have strong hydrogen bonding interactions and electrostatic interactions with Ap6A. In addition, we further find that three residues, Ser38, Leu39 and Glu46, coordinate enzyme-bound Mg2+ ions in complex N-A. The Glu46 is consistent with the experimental results by Iwai et al., and the other four residues mentioned above may also play vital roles in catalysis of Ndx1.