The expression of exogenous DNA in Sertoli cells is essential for studying its functional genomics, pathway analysis, and medical applications. Electroporation is a valuable tool for nucleic acid delivery, even in primarily cultured cells, which are considered difficult to transfect. In this study, we developed an optimized protocol for electroporation-based transfection of Sertoli cells and compared its efficiency with conventional lipofection. Sertoli cells were transfected with pCMV-GFP plasmid by square-wave electroporation under different conditions. After transfection of plasmid into Sertoli cells, enhanced green fluorescent protein (EGFP) expression could be easily detected by fluorescent microscopy, and cell survival was evaluated by dye exclusion assay using Trypan blue. In terms of both cell survival and the percentage expressing EGFP, 250 V was determined to produce the greatest number of transiently transfected cells. Keeping the voltage constant (250 V), relatively high cell survival (76.5% ± 3.4%) and transfection efficiency (30.6% ± 5.6%) were observed with a pulse length of 20 μm. The number of pulses significantly affected cell survival and EGFP expression (P < 0.001). Cell survival clearly decreased following one to three pulses, from 83.9% ± 6.1% to 3.2% ± 1.1%, with EGFP expression increasing from 41.8% ± 9.4% to 66.7% ± 5.2%. The yield of positive cells increased with increasing concentration of plasmid DNA (range, 10–50 μg/ml), from 14.0% ± 2.8% to 35.0% ± 6.3%, but cell viability steadily decreased following 20 μg/ml plasmid DNA, from 73.1% ± 4.9% to 57.0% ± 6.6%. Compared with two popular cationic lipid transfection methods, the transfection efficiency of electroporation (21.5% ± 5.7%) was significantly higher than those of Lipofectamine 2000 (2.9% ± 1.0%) and Effectene (1.9% ± 0.8%) in this experiment (P < 0.001). We describe the process of optimizing electroporation conditions, and the successful electroporation of plasmid DNA into primarily cultured Sertoli cells. Our results indicate that the method of electroporation is more suitable than other approaches for the transfection of Sertoli cells.