We propose a mechanism for DNA release from lipoplexes in cells that accounts for various observations of lipoplex-anionic lipid interactions. We examined the structural evolution of lipoplexes upon interaction with cellular lipids by synchrotron small-angle X-ray diffraction (SAXD), and the extent of DNA release from lipoplexes was determined by gel electrophoresis. We find that the interaction of lipoplexes with anionic cellular lipids is a two-stage process. In the first step, anionic lipids laterally diffuse into the complex and neutralize the cationic lipids. As a result, the membrane charge density of lipoplexes decreases and interactions between cationic lipids and DNA become weaker, but DNA is extremely poorly released. Only after the cationic charge of lipoplex membranes is completely neutralized by anionic lipids does DNA starts to be released significantly.