We have previously reported that mannosylated cationic liposome consisting with the mannosylated cationic cholesterol derivative Man-C4-Chol (Man) and dioleoylphosphatidylethanolamine (DOPE) (Man/DOPE) could deliver DNA to the liver by intravenous administration via mannose receptor-mediated endocytosis, however, rapid degradation in lysosomes might be a rate-limiting step in its gene transfection. In this study, we tried to evaluate the role of DOPE in in vivo gene transfer by comparing its transfection efficacy with mannosylated liposomes composed of Man and dioleoylphosphatidylcholine (DOPC) (Man/DOPC). In vitro studies showed that the cellular association of both liposome/pCMV-Luc complexes was almost the same, although Man/DOPE complex showed about 10-fold higher transfection activity than Man/DOPC complex. After intraportal administration into mice, Man/DOPE complex showed higher gene expression than Man/DOPC complex, suggesting that DOPE improves intracellular trafficking in target cells under in vivo conditions. An intravenous administration study demonstrated that Man/DOPE complex was accumulated in the liver more efficiently and achieved a higher gene expression in the liver than Man/DOPC complex. Thus, we conclude that the property of DOPE in mannosylated liposomes contributes to the efficient gene expression in the target site through enhanced distribution to the target site and intracellular sorting in the target cells under in vivo conditions.