A novel electroporation system was developed to introduce transient membrane pores to cells in a spatially and temporally controlled manner, allowing us to achieve fast electrotransfection and live cell staining as well as to systematically interrogate the dynamics of the cell membrane. Specifically, using this platform, we showed that both reversible and irreversible electroporation could be induced in the cell population, with nano-sized membrane pores in the former case being able to self-reseal in ~10 min. In addition, green fluorescent protein(GFP)-vinculin plasmid and 543 phalloidin have been delivered successively into fibroblast cells, which enables us to monitor the distinct roles of vinculin and F-actin in cell adhesion and migration as well as their possible interplay during these processes. Compared to conventional bulk electroporation and staining methods, the new system offers advantages such as low-voltage operation, cellular level manipulation and testing, fast and adjustable transfection/staining and real-time monitoring; the new system therefore could be useful in different biophysical studies in the future.