Naked plasmid DNA (pDNA)-based gene therapy has low delivery efficiency, and consequently, low therapeutic effect. We present a biodegradable nonionic triblock copolymer, PEG(13)-PLGA(10)-PEG(13), to enhance gene delivery efficiency in skeletal muscle. Effects of PEG(13)-PLGA(10)-PEG(13) on physicochemical properties of pDNA were evaluated by atomic force microscopy (AFM) imaging, gel electrophoresis and zeta-potential analysis. AFM imaging suggested a slightly compacted structure of pDNA when it was mixed with the polymer, while zeta-potential measurement indicated an increased surface potential of negatively charged pDNA. PEG(13)-PLGA(10)-PEG(13) showed a relatively lower toxicity compared to Pluronic P85 in a skeletal muscle cell line. The luciferase expression of pDNA delivered in 0.25% polymer solution was up to three orders of magnitude more than branched polyethylenimine (bPEI(25 k))/pDNA and three times more than that of naked pDNA five days after intramuscular administration. This in vivo gene delivery enhancement was also observed displaying a two-fold higher expression of human vascular endothelial growth factor (VEGF). Based on fluorescence labeled pDNA distribution, it is speculated that the greater diffusivity of PEG(13)-PLGA(10)-PEG(13)/pDNA compared to bPEI(25 k)/pDNA accounts for better transfection efficiency in vivo. To summarize, combining PEG(13)-PLGA(10)-PEG(13) with pDNA possesses the potential to improve gene delivery efficiency in skeletal muscle.