Disc-shaped bicelles are formed by mixing long-chain lipids with short-chain lipids at suitable molar ratios and they have a relatively uniform size, typically around a few tens of nanometers in diameter. Different from the typically formulated cationic or anionic liposome–DNA complexes, which are used as nonviral vectors for improving the transfection efficiency of gene therapy, a novel way of packing the DNA can be developed by using the much smaller disc-like bicelles. We demonstrate that anionic lipid bicelle-ion–DNA (AB–DNA) complexes can be formed with the help of divalent ions. Multi-stacked AB–DNA complexes can be formed with diameters of around 50–100 nm and lengths of around 50–150 nm as revealed by TEM. Using the anionic lipid–DNA complexes has the advantage of lower cytotoxicity than using cationic lipids. The interaction of DNA with anionic bicelles was investigated by SAXS. It was found that the anionic bicelle could not form stable complexes with DNA at low calcium ion concentrations, such as 1 mM. The AB–DNA complexes can be formed in the investigated range of 10 mM to 100 mM calcium ion concentrations. However, for an equal anionic lipid charge and DNA charge system, an ion-membrane phase (multilamellar vesicles) would gradually appear as the calcium ion concentration is increased above a critical concentration. It indicates that DNA could be packed closer at above the critical divalent ion concentration. If more DNA is added to such a two-phase coexistence system (originally with the total anionic lipid charge equal to that of DNA), the ion-membrane phase could be transformed into the AB–DNA complexes. As a result, more DNA can be packed in the form of AB–DNA complexes at above the critical calcium ion concentration.