Amphiphilic lipids are promising for efficient intracellular delivery of nucleic acids. In this study, two new amphiphilic carriers, EKHCO and EHHKCO, were designed and synthesized as multifunctional carriers for efficient intracellular delivery of nucleic acids. The critical micelle concentrations of EKHCO and EHHKCO were 9.50 and 6.87 mu M, respectively. Dynamic light scattering showed that the surfactants complexed with plasmid DNA and si RNA to form stable nanoparticles at the concentrations below their critical micelle concentrations. The nanoparticles of the surfactants with pDNA and si RNA exhibited pH-sensitive hemolysis against rat red blood cells when the pH decreased from 7.4 to 5.5, the endosomal-lysosomal pH. The nanoparticles of EHHKCO showed more concentration-dependent pH sensitivity than those of EKHCO. The EHHKCO and EKHCO nanoparticles of both pNDA and siRNA exhibited low cytotoxicity of at physiological pH. Both EKHCO and EHHKCO resulted in high intracellular uptake of pDNA and siRNA. EKHCO and EHHKCO resulted in relatively lower luciferase expression efficiency in U87 cells than DOTAP but produced a much higher percentage of GFP expression in the transfected cells than DOTAP. Both EKHCO and EHHKCO mediated much higher gene silencing efficiency of luciferase and green fluorescence protein (GFP) than DOTAP. The surfactants were more effective for intracellular siRNA delivery than intracellular delivery of plasmid DNA. The pH-sensitive amphiphilic carriers are promising multifunctional carriers for intracellular delivery of nucleic acids.