Genetic engineering approaches to improve therapeutic potentials of mesenchymal stem cells (MSCs) have been made by viral or non-viral gene delivery methods. Viral methods have severe limitations in clinical application due to potential oncogenic, pathogenic, and immunogenic risks while non-viral ones have suffered from low transfection efficiency and transient weak expression as MSCs are hard-to-transfect cells. In this study, minicircle, which is a minimal expression vector free of bacterial sequences, was employed for MSC transfection as a non-viral gene delivery. Conventional cationic liposome method was not effective for MSC transfection to result in very low transfection efficiency (less than 5%). Microporation, a new electroporation method, greatly improved the transfection efficiency of minicircles up to 66% in MSCs without significant loss of cell viability. Furthermore, minicircle microporation generated much stronger and prolonged transgene expression, compared to plasmid microporation. When the MSCs transfected to express firefly luciferase by minicircle microporation were subcutaneously injected to mice, their bioluminescence was monitored for more than a week whereas the bioluminescence of the MSCs induced by plasmid microporation rapidly decreased and disappeared in mice within three days. By using minicircle microporation as a non-viral gene delivery, the engineered MSCs to overexpress CXCR4 were shown to have greatly increased homing ability toward the injury site through in vivo bioluminescence imaging in mice, which showed a promise to enhance therapeutic potentials of MSCs in clinical applications.