The objective of this study was to study the transfection efficiency of quaternized N-(4-pyridinylmethyl) chitosan; TM-Py-CS, using the pDNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh 7 cells). The factors affecting the transfection efficiency, e.g. degree of quaternization (DQ), the extent of N-pyridinylmethyl substitution (ES) and weight ratio, have been investigated. The results revealed that TM-Py-CS was able to condense with pDNA. Illustrated by agarose gel electrophoresis, complete complexes of TM69Py62CS/DNA were formed at weight ratio above 1.1, whereas those of TM53Py40CS/DNA and TM52Py13CS/DNA were above 1.8 and 8, respectively. TM69Py62CS showed superior transfection efficiency to TM53Py40CS, TM52Py13CS, TM65CS and TM43CS at all weight ratios tested. The highest transfection efficiency of TM69Py62CS/DNA complexes was found at weight ratio of 4. The results indicated that the improved gene transfection was possibly due to 4-pyridinylmethyl substitution on CS which promoted the interaction and condensation with DNA as well as N-quaternization which increased CS water solubility. In cytotoxicity studies, high concentration of TM-Py-CS and TM-CS could decrease the Huh 7 cell viability. In conclusion, this novel CS derivative, TM69Py62CS, showed promising potential as a gene carrier by efficient DNA condensation