Ionic liquids (ILs) have been extensively used for stabilization and long-term DNA storage. However, molecular level understanding of the role of the hydrogen bond of DNA with ILs in its stabilization is still inadequate. Two ILs, namely, 1,1,3,3-tetramethylguanidinium acetate (TMG) and 2,2-diethyl-1,1,3,3-tetramethylguanidinium acetate (DETMG), have been synthesized, of which TMG has a hydrogen bonding N–H group whereas DETMG does not contain any hydrogen bonding site. It has been found that both TMG and DETMG cations interact in the groove region of DNA; however, their mode of interaction is distinctly different, which causes the stabilization of DNA in the presence of TMG, whereas the effect is opposite in the case of DETMG. It is apparent from the data that only the accommodation of ILs in the groove region is not enough for the stabilization of DNA. MD simulation and spectroscopic studies combinedly indicate that the hydrogen bonding capability of the TMG cation enhances the hydrogen bonding between the Watson–Crick base pairs of DNA, resulting in its stabilization. In contrast, the bigger size as well as the absence of the hydrogen bonding site of the DETMG cation perturbs the minor groove width and base pair step parameters of DNA during its intrusion into the minor groove, which decreases the hydrogen bond between the Watson–Crick base pairs of DNA, leading to destabilization.