Synthetic materials that assemble reversibly with polyanions under physiological conditions are of interest for a broad range of biotechnical applications. Cationic polymers are used widely as agents for the condensation of DNA, but polycations also introduce practical limitations in applications for which subsequent dissociation or disassembly of polycation/DNA complexes is desired. The design of cationic polymers that promote the release of associated DNA presents a challenge because it requires the introduction of functionality that is inherently opposed to that required for efficient DNA condensation. Here, we report the synthesis and biophysical characterization of linear poly(ethylenimine) (LPEI) functionalized with methyl ester side chains. The gradual hydrolysis of the ester functionality in these materials under physiologically relevant conditions results in a controlled reduction in cationic charge density and a change in the nature of the electrostatic interactions between the polymers and plasmid DNA, as determined by agarose gel electrophoresis. Using this approach, it is possible to mediate the dissociation of DNA from polymer over a period of hours to days by varying the mole percentage of methyl esters incorporated into the polymer. Polymers having a high degree of substitution (e.g., 80 or 100 mol %) release DNA more rapidly than less-substituted polymers (e.g., 40 and 60 mol % functionalized). Polymers having 20 mol % substitution did not release DNA in these experiments. These charge-shifting materials could provide a basis for the design and fabrication of polyelectrolyte complexes and assemblies that sustain the release of DNA in solution and at interfaces.