The description of the phosphate group and its reactions with nitrogen species appears to be challenging using semi-empirical quantum chemical methods, and this holds for DFTB3 too. A new parameterization of DFTB3, consisting of a new P–N repulsive function, has been developed to improve its performance for reactions in which a P–N bond is replaced by a P–O bond or vice versa. Extended-sampling QM/MM simulations using the new parameterization of DFTB3 represent biochemical phosphorylation and hydrolysis reactions involving P–N bonds accurately. The parameter set is benchmarked on a reaction modeling the autophosphorylation of histidine, and is applied to study the complex mechanism of the acidic hydrolysis of an anticancer drug, as well as to the autophosphorylation of a genuine histidine kinase protein.