Several barriers need to be overcome to ensure successful gene transfection, including passing of the foreign gene through the plasma membrane, escape of this material from lysosomal degradation, and its translocation into the nucleus. We previously showed that the biosurfactant mannosylerythritol lipid-A (MEL-A) enhanced the efficiency of gene transfection mediated by cationic liposomes by facilitating rapid delivery of foreign genes into target cells through membrane fusion between liposomes and the plasma membrane. Moreover, using MEL-A-containing cationic liposomes, the foreign gene was efficiently delivered into the nucleus because it was released directly into the cytosol and thus escaped lysosomal degradation. Here we investigated the effect of pre-condensation of plasmid DNA by a cationic polymer, protamine, on gene transfection. We found that the efficiency of pre-condensed DNA transfection mediated by MEL-A-containing OH liposomes was >10 times higher than that of non-condensed DNA transfection. In contrast, the efficiency of pre-condensed DNA transfection mediated by OH liposomes was only 1.5 times higher than that of non-condensed DNA transfection. MEL-A did not influence plasmid DNA encapsulation by cationic liposomes, but it greatly accelerated the nuclear delivery of pre-condensed plasmid DNA. Our findings indicate that MEL-A and protamine synergistically accelerate the nuclear delivery of foreign gene and consequently promote gene transfection efficiency.