Under physiological conditions, G-C base pairs are more stable than A-T base pairs. In a previous study, we showed that in the hydrated ionic liquid of choline dihydrogen phosphate, the stabilities of these base pairs are reversed. In the present study, we elucidated the unique binding interactions of choline ions with DNA atoms from a microscopic viewpoint using molecular dynamics simulations. Three times more choline ions bind to the DNA duplex than sodium ions. Sodium ions bind closely but not stably; in contrast, the choline ions bind through multiple hydrogen bonding networks with DNA atoms stably. The affinity of choline ion for the minor groove of A-T base pairs is more than 2 times that for other groove areas. In the narrow A-T minor groove, choline ion has high affinity for the ribose atoms of thymine. Choline ions also destabilize the formation of hydrogen bonds between G-C base pairs by binding to base atoms preferentially for both of duplex and single-strand DNA, which are associated with the bonds between G-C base pairs. Our new finding will not only lead to better control of DNA stability for use in DNA nanodevices, but also provide new insight into the stability of DNA duplexes under crowding conditions found in living cells.