Genetically modified mesenchymal stem cells (MSCs) have great potential in the application of regenerative medicine and molecular therapy. In the present manuscript, we introduce a nanopolymer, polyethylenimine600-β-cyclodextrin (PEI600-β-CyD), as an efficient polyplex-forming plasmid delivery agent with low toxicity and ideal transfection efficiency on primary MSCs. PEI600-β-CyD causes significantly less cytotoxicity and apoptosis on MSCs than 25 kDa high-molecular-weight PEI (PEI25kDa). PEI600-β-CyD also exhibits similar transfection efficiency as PEI25kDa on MSCs, which is higher than that of PEI600Da. Quantum dot-labeled plasmids show that PEI600-β-CyD or PEI25kDa delivers the plasmids in a more scattered manner than PEI600Da does. Further study shows that PEI600-β-CyD and PEI25kDa are more capable of delivering plasmids into the cell lysosome and nucleus than PEI600Da, which correlates well with the results of their transfection-efficiency assay. Moreover, among the three vectors, PEI600-β-CyD has the most capacity of enhancing the alkaline phosphatase activity of MSCs by transfecting bone morphogenetic protein 2, 7, or special AT-rich sequence-binding protein 2. These results clearly indicate that PEI600-β-CyD is a safe and effective candidate for the nonviral gene delivery of MSCs because of its ideal inclusion ability and proton sponge effect, and the application of this nanopolymer warrants further investigation.