Techniques used for nonviral gene transfection often have poor spatial resolution. In this letter, we present a microelectrode array (MEA) system that can precisely transfect exogenous molecules into targeted primary neurons using microelectroporation. An optimal cathodic pulse 4 V in amplitude and 1 ms in duration resulted in a transfection efficiency of 56% and a viability of 82%. Finally, siRNA molecules were transfected into targeted neurons in culture using the aforementioned system.