A major goal for gene therapy is to obtain targeted vectors that transfer genes efficiently to specific cell types. The liver possesses a variety of characteristics that make this organ very attractive for gene therapy. In the present study, four cholesterylated thiogalactosides 1a approximately d with different spacer length were synthesized to formulate novel lipid-polycation-DNA (LPD) complexes, which were composed of galactosylated cationic liposomes, protamine sulfate and plasmid DNA. The galactosylated LPD1c significantly improved the levels of gene expression in cultured hepatoma cells HepG2 and SMMC-7721, while LPD1a and LPD1b did not significantly improve the levels compared with non-galactosylated LPD. Meanwhile, increased transfection activity was not observed in mouse fibroblasts L929 for galactosylated LPDs. Cytotoxicity of galactosylated LPDs assay showed they had no obvious toxicities to L929 cells and HepG2 cells. In summary, the length of the spacer between the anchor and galactose residues was important for the recognition of asialoglycoprotein receptor. The LPD1c described here, combining the condensing effect of protamine and the targeting capability of cholesterylated thiogalactosides, are potentially useful gene carriers to liver parenchymal cells.