In this work, the influence of the metal ions present in vertebrate Sonic Hedgehog was assessed by a series of molecular mechanics molecular dynamics simulations with differing ionic compositions. The obtained data suggest that Ca(II) binding has a very distinct influence on the composition of the protein surface surrounding the binding site by shaping several ionic interactions with negatively charged sidechains that otherwise would be pointing towards the solvent, repelling potential ligands. Furthermore, the Ca(II) ions play an important role in the stability of the loop regions where they are coordinated. In contrast, the removal of the Zn(II) ion results in no noticeable destabilization of its chemical surrounding, however, it is shown that the destabilizing effect of removed Ca(II) ions is amplified if Zn(II) is absent as well. Furthermore, a quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulation of Sonic Hedgehog with special focus on the Zn(II) binding site has been conducted. The results indicate that QM/MM in contrast to pure MM accurately reproduces structural features also found by experimental studies and therefore is able to provide credible predictions not only of the dynamical properties of the studied system but also of protein–ligand interactions at the metal ion binding site.