In this study, we examined the use of polyethyleneimine (PEI) as a carrier for gene delivery in human adipose tissue-derived stem cells (hADSCs). These multipotent cells can form bone, cartilage, adipose, and other connective tissues. In primary culture, hADSCs are fibroblastic in appearance in primary culture, and they show a high rate of proliferation for at least five passages. Immunophenotyping showed that these cells are positive for the mesenchymal stem cell markers CD29 and CD44 but negative for the hematopoietic cell surface markers CD34, CD45, and c-kit. PEI and Lipofectamine were compared as gene carriers for hADSCs. DNA completely bound PEI at a negative-to-positive (N/P) charge ratio of 4. The PEI-DNA complexes were spherical with smooth surfaces. As the proportion of PEI was increased, the size of the PEI-DNA complexes decreased from 990 to 130nm, the positive surface charge decreased, and the cytotoxicity increased. Flow cytometry revealed that the transfection efficiency using PEI at N/P charge ratios of 4 and 8 was higher than that of Lipofectamine. The highest transfection efficiency (19%) was obtained at an N/P charge ratio of 8. After transfection, the enhanced green fluorescent protein (EGFP) started to localize in the nuclei of hADSCs at 4h 30m and localize over cytoplasm from 9h 30m. In conclusion, PEI acts as an effective gene carrier for hADSCs.