Current challenges in embryonic-stem cell (ESC) research include the inability of sustaining and culturing of undifferentiated ESCs over time. Growth-arrested feeder cells are essential to the culture and sustaining of undifferentiated ESCs, and they are currently prepared using gamma-radiation and chemical inactivation. Both techniques have severe limitations. In this study, we developed a new, simple and effective technique (pulsed electric fields, PEFs) to produce viable growth-arrested cells (RTS34st) and used them as high-quality feeder cells to culture and sustain undifferentiated zebrafish ESCs over time. The cells were exposed to 25 sequential 10-ns electric pulses (10nsEPs) of 25, 40 and 150 kV/cm with 1-s pulse interval, or 2 sequential 50-mus electric pulses (50microsEPs) of 2.83, 1.78 and 0.78 kV/cm with 5-s pulse interval, respectively. We found that the cellular effects of PEFs depended directly upon the duration, number and electric field strength of the pulses, showing the feasibility of tuning them to produce various types of growth-arrested cells for culturing undifferentiated ESCs. Both 10nsEPs of 40 kV/cm produced by a 10nsEP generator and 50microsEPs of 1.78 kV/cm provided by inexpensive and widely available conventional electroporators, generated high-quality growth-arrested feeder cells for proliferation of undifferentiated ESCs over time. PEFs can therefore be used to replace radiation and chemical inactivation methods for preparation of growth-arrested feeder cells for advancing ESC research.