Folate conjugated N-trimethyl chitosan (folate-TMC) that was used for intracellular delivery of protein before was studied as a gene delivery carrier in this study using N-trimethyl chitosan (TMC) as a reference. MTT assay indicated that the two polymers were much less toxic than PEI. Agarose gel electrophoresis indicated that the two polymers effectively condensed pDNA. TMC/pDNA complex and folate-TMC/pDNA complex were nano-scale spherical particles confirmed by atomic force microscopy. Cellular uptake of the folate-TMC/pDNA complex containing YOYO-1 labeled pDNA in KB cells was enhanced compared with that of the TMC/pDNA complex and was inhibited by free folate (1 mM) in the medium. Transfection efficiency of the folate-TMC/pDNA complex in KB cells and SKOV3 cells (folate receptor over-expressing cell lines) increased with increasing N/P ratio and were enhanced up to 1.6-fold and 1.4-fold compared with those of the TMC/pDNA complexes, however, there was no significant difference between transfection efficiencies of the two complexes in A549 cells and NIH/3T3 cells (folate receptor deficient cell lines). It was concluded that the increase in transfection efficiencies of the folate-TMC/pDNA complexes were attributed to folate receptor mediated endocytosis. Subcellular distributions of both of the complexes at different time points in the process of cellular uptake were examined by confocal laser scanning microscope, which suggested that different intracellular trafficking pathways were employed by the two complexes.