Cationic lipoplexes, which consist of cationic lipids and DNA, are widely used in gene transfection. Recently, some reports suggest that the transfection efficiency of lipoplexes is affected by its formulation and physicochemical properties. To improve the lipoplex transfection efficiency, we examined gene transfection using lipoplexes treated with high hydrostatic pressure (HHP) that affects the phase behavior of the phospholipid bilayer. Lipoplexes were prepared with different cationic lipid to DNA ratios and then subjected to hydrostatic pressure at 5,000 and 10,000 atm. Non-pressurized lipoplex and HHP-treated lipoplex (after removing pressure) were evaluated. Dynamic light scattering measurement showed that the size of HHP-treated lipoplex increased compared with non-pressurized lipoplex, suggesting that formulation change and assembly of lipoplexes occurred under high hydrostatic pressure. Lipoplexes with and without HHP treatment were transferred into HeLa and COS-7 cells. The transfection efficiency of HHP-treated lipoplex increased compared with non-pressurized lipoplex, irrespective of the mixture ratio. Also, the transfection efficiency of HHP-treated lipoplex remained higher for several days. These results suggest that high hydrostatic pressure induces structural change in lipoplex, thereby affecting the transfection efficiency.