Beryllium fluoride is widely used as a phosphoryl analogue in macromolecular studies, which are not only fluoride-sensitive but also magnesium-dependent. The beryllium fluorides are a mixture of different species including BeF3- and BeF42- exchanging under thermodynamic equilibrium in neutral aqueous solutions. In the cases of mimicking phosphate group transfer, both beryllium fluoride and the magnesium ion are generally needed. However, the impact of magnesium on the bioactivity of beryllium fluoride is not clear. We have found by F-19 NMR spectroscopy that Mg2+ can severely affect the chemical exchange kinetics between BeF3- and BeF42-. When the F- concentration is relatively low, the presence of 10.0 mM Mg2+ can accelerate the exchange rate 3-4 fold. However, when the F- concentration is relatively high, the Mg2+ effect on the chemical exchange vanishes. On the basis of these findings, we proposed a possible mechanism that BeF42- and Mg2+ form an ion pair that affects the distribution of beryllium fluoride species and thus the activity in the solution.