Introduction of various kinds of exogenous genes is an important step for control of differentiation in stem cell biology and regenerative medicine. However, some kinds of cells are vulnerable to manipulations such as gene delivery. In this context, a gene introduction method with higher efficiency and safety is required. Bone marrow stromal cells (BMSCs) offer possibilities for clinical application because of their potential for expandability and ability to be auto-transplanted. In this study, we established an efficient induction system of dopamine-producing neuronal cells from BMSCs in several species using the spermine-pullulan-mediated reverse transfection technique. In this system, introduced exogenous plasmid genes were successfully transcribed and expressed as proteins in the cytoplasm of BMSCs with the smallest number of cell death. Microtubule-associated protein 2 and anti-beta-tubulin class III+ neurons were successfully delivered from human, monkey, and mouse BMSCs, and further treatment with trophic factors promoted differentiation of induced neuronal cells into dopamine-producing cells that were positive for tyrosine hydroxylase and secreted dopamine after high K+ stimulation in high-performance liquid chromatography analysis. Our study indicates the availability of the reverse transfection method for the induction of dopamine-producing neuronal cells from BMSCs, which is expected to apply to cell-based therapy in Parkinson's disease.