Significant effort has been made to develop synthetic metal complexes that hydrolyze DNA. Here we report a new dicerium complex, Ce2(HXTA) (HXTA = 5-methyl-2-hydroxy-l,3-xylene-α,α-diamine-N,N,N′, N′-tetraacetic acid), which can hydrolyze DNA at pH 8 and 37°C. This complex hydrolyzes DNA restriction fragments to give products with high regioselectivity, affording >90% 5′-OPO3 and 3′-OH ends, like the products of DNA hydrolyzing enzymes. Ce2(HXTA) also hydrolyzes Litmus 29 plasmid DNA to afford both nicked and linear DNA. Analysis of the relative amounts of supercoiled, nicked, and linear DNA present show that there is one double-strand cleavage per ten single-strand cleavages, indicating that the linear DNA formed cannot be the result of two random single-strand cleavage events. The kinetics of nicked and linear DNA formation are comparable, both being associated with apparent first-order rate constants of approximately 1 × 10-4 s-1 for complex concentrations of 10-5-10-4 M. These observations suggest that similar factors affect the hydrolysis of the first and second DNA strands and that cleaving the phosphodiester bond is likely the rate determining step in both cases. This is the first detailed study of a metal complex shown to mimic DNA hydrolases in their capability to effect double-strand DNA hydrolysis regioselectively at the 3′-O-P bond.