Electroporation is a safe and efficient method to transfect human tooth germ stem cells (hTGSCs), which are mainly mesenchymal stem cells (MSCs). In this study, pluronic F68 (PF68), a nonionic and low-foaming surfactant, was used for the first time to increase the transfection efficiency in electroporation of hTGSCs. Our results revealed that when PF68 was used in the preincubation, electroporation, and recovery medium, it dramatically increased transfection efficiency. Moreover, use of PF68 did not alter the differentiation capacity of hTGSCs, whereas cells electroporated without PF68 had significantly lower expression of Oct-4, pluripotency marker gene, and alkaline phosphatase (ALP) activity upon osteogenic differentiation. We found that PF68 increases transfection efficiency in both electroporation and chemical transfection of cancer cells lines MCF-7 and HeLa. The study suggested that PF68 might be a safe and reliable chemical to use for increasing transfection efficiency during electroporation and chemical transfections.