The DNase1 family of enzymes are endonucleases that are important in the removal of dsDNA debris. Null functioning mutations in DNase1 and DNase1L3 result in the development of systemic lupus erythematosus and related autoimmune conditions. Interestingly, despite the ubiquity of DNase1 and related enzymes, the catalytic mechanism of this family of nucleases has not been fully explored. Using X-ray crystallography we have solved the structure of recombinant human DNase1L3. Careful analysis of our results revealed a second magnesium ion coordinated in the active site of DNase1L3. The experimental structures for the DNase1 family were treated using full-atom molecular dynamics to visualize the cation coordination of DNase1 and DNase1L3. Molecular dynamics was then used to generate an initial structure for hybrid quantum mechanics/molecular mechanics simulation. QM/MM simulation enabled an accurate investigation of the electronic structure of the active site of DNase1L3 and DNase1. Using QM/MM methods, the free energy barrier for the double-divalent phosphodiester reaction mechanism was calculated. The calculated reaction rates for DNase1 and DNase1L3 were then compared to experimentally determined rates to support the method of analysis. This investigation has thoroughly investigated the catalytic mechanism of the DNase1 family to clarify the lowest energy pathway amongst previously proposed mechanisms.