The delivery of genetic material to cells offers the potential to treat many genetic diseases. Cationic polymers, specifically poly(ethylene imine) (PEI), are promising gene delivery vectors due to their inherent ability to condense genetic material and successfully affect its transfection. However, PEI and many other cationic polymers also exhibit high cytotoxicity. To systematically study the effect of polymer architecture on gene delivery efficiency and cell cytotoxicity, a set of cyclic PEIs were prepared for the first time and compared to a set of linear PEIs of the exact same molecular weight. Subsequent in vitro transfection studies determined a higher transfection efficiency for each cyclic PEI sample when compared to its linear PEI analogue in addition to reduced toxicity relative to the branched PEI "gold standard" control. These results highlight the critical role that the architecture of PEI can play in both optimizing transfection and reducing cell toxicity.