We have investigated the effect of serum on nanometric structure, size, surface potential, DNA-binding capacity, and transfection efficiency of DDAB-DOPE/DNA and DC-Chol-DOPE/DNA lipoplexes as a function of membrane charge density and cationic lipid/DNA charge ratio. In the absence of serum, the nanometric structure and DNA binding capacity of lipoplexes determined the transfection efficiency. When serum was added, the transfection efficiency of all lipoplex formulations was found to increase. We identified structural stability and an increase in size in serum as major parameters regulating the efficiency of lipofection. By extrapolation, we propose that serum, regulating the size of resistant lipid-DNA complexes, can control the mechanism of internalization of lipoplexes and, in turn, their efficiency.