The objective of this study was to investigate the transfection efficiency of quaternized N-(4-N,N-dimethylaminobenzyl) chitosan, TM47-Bz42-CS, using the plasmid DNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh7 cells), in comparison to quaternized chitosan (TM43-CS) and chitosan (CS). Factors affecting the transfection efficiency, such as the carrier/DNA weight ratio, the pH of the culture medium, and the presence of serum, have been investigated. The results revealed that TM47-Bz42-CS was able to condense with pDNA. As illustrated by the agarose gel electrophoresis, the complete complexes of TM47-Bz42-CS/DNA were formed at a weight ratio of above 0.5, whereas those of TM43-CS/DNA and CS/DNA were formed at a ratio of above 1. TM47-Bz42-CS showed superior transfection efficiency to TM43-CS and CS at all weight ratios tested. Higher transfection efficiency and gene expression were observed when the carrier/DNA weight ratios increased. The highest transfection efficiency was found at a weight ratio of 8. The results indicated that the improved gene transfection was due to the hydrophobic group (N,N-dimethylaminobenzyl) substitution on CS, which promoted the interaction and condensation with DNA, as well as N-quaternization, which increased the CS water solubility. During cytotoxicity studies, it was found that high concentrations of TM47-Bz42-CS and TM43-CS could decrease the Huh7 cell viability. In conclusion, this novel CS derivative, TM47-Bz42-CS, shows promising potential as a gene carrier by efficient DNA condensation and a mediated higher level of gene transfection in Huh7 cells.