In this study, hydrophilic PLGA/Pluronic F127 scaffolds loaded with a pDNA/PEI-PEG complex were prepared to estimate their potential use as a polymeric matrix for pDNA delivery. The scaffold was fabricated by a novel precipitation/particulate leaching method. The prepared pDNA/PEI-PEG complex-loaded PLGA/Pluronic F127 scaffold exhibited a highly porous (porosity, 93–95%) and open pore structure, as well as hydrophilicity, which can provide the good environment for cell adhesion and growth. The pDNA/PEI-PEG complexes were efficiently loaded into the PLGA/Pluronic F127 scaffold and continuously released from the scaffolds up to ∼90% of the initial loading amount over a period of 8 wk, which may lead to continuous gene transfection into human bone marrow mesenchymal stem cells (hBMMSCs). From the in vitro cell culture in the scaffolds for transfection, it was observed that the pDNA/PEI-PEG complex-loaded hydrophilic PLGA/Pluronic F127 scaffold has a higher transfection efficiency of the pDNA/PEI-PEG complexes into hBMMSCs than the hydrophobic PLGA ones. The cell viability associated with the pDNA/PEI-PEG complexes released from the PLGA/Pluronic F127 scaffold was not significantly different from that of the PLGA/Pluronic F127 scaffold without pDNA, indicating its low cytotoxicity, probably due to the sustained release of the pDNA/PEI-PEG complex from the scaffolds. From these results, we could suggest that the pDNA/PEI-PEG complex-loaded hydrophilic PLGA/Pluronic F127 scaffold can be an effective gene delivery system for 3D tissue formation. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 2011.