Background: Dermal fibroblast is a powerful tool for the study of ex vivo DNA delivery in development of both cell therapy and tissue engineering products. Using genetic modification, fibroblasts can be diversely adapted and made suitable for clinical gene therapy. In this study, we first compared several non-viral transfection methods including nucleofection in rat and human primary dermal fibroblast. In addition, the original protocol for nucleofection of primary mammalian fibroblasts was modified in order to achieve the highest possible transfection efficiency, as determined by flow cytometry analysis of the green fluorescent protein (GFP) expression. Results: the results showed that transfection performance of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Calf Serum (FCS) yielded the best transfection efficiency with rat dermal fibroblasts and ITS (insulin, transferrin, and sodium selenite solution) was comparable to the standard nucleofection solution for human dermal fibroblasts. Conclusion: Our results suggest a promising application of the modified nucleofection method for GMP compatible therapeutic translational medical research.