Cationic lipids are promising candidates as transporters of genetic material into cells. Therefore, detailed knowledge on the molecular interaction between polynucleotides (DNA) and cationic lipids is needed to improve our understanding of these so-called lipoplexes. Here, for the first time, we have investigated the role of water in DNA-lipid interaction using vibrational sum-frequency generation (VSFG). We show that the DNA-lipid complexation induces a dramatic restructuring of interfacial water, with less than one monolayer of water remaining between the DNA and the lipid; we also observe that the lipid-bound water reorients upon interaction with DNA. This is apparent from the rather dramatic changes in the vibrational response of interfacial water. The approach presented here has a very high (picomolar) sensitivity and allows for the quantification of the DNA-lipid association constant.