Polyethylenimine (PEI) is one of the most extensively studied non-viral vectors but its cytotoxicity limits its clinical value. PLGA nanospheres are biocompatible and can facilitate sustained release of plasmid DNA. This study compares the cytotoxicity and long-term transgene expression between PLGA nanosphere and PEI. PLGA nanospheres were significantly less cytotoxic than PEI at various concentrations. PLGA nanospheres induced significantly higher transgene expression in vitro for a longer duration (21 days) than PEI. We conclude that PLGA nanospheres have potential as gene delivery vehicles for use in gene therapy for diseases in which a long-term therapeutic gene expression regimen is necessary.