Amphiphilic imidazolium-based ionic liquids (ILs) have proven their efficacy in altering the membrane integrity and dynamics. The present article investigates the phase-separated domains in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membrane induced by 1,3 dialkylated imidazolium IL. Isotherm measurements on DPPC monolayers formed at the air-water interface have shown a decrease in the mean molecular area with the addition of this IL. The positive value of the excess Gibbs free energy of mixing indicates an unfavorable mixing of the IL into the lipid. This leads to IL-induced phase-separated domains in the multilayer of the lipid confirmed by the occurrence of two sets of equidistance peaks in the X-ray reflectivity data. The electron density profile along the surface normal obtained by the swelling method shows the bilayer thickness of the newly formed IL-rich phase to be substantially lower (∼34 Å) than the DPPC phase (∼45.8 Å). This IL-rich phase has been confirmed to be interdigitated, showing an enhanced electron density in the tail region due to the overlapping hydrocarbon chains. Differential scanning calorimetry measurements showed that the incorporation of IL enhances the fluidity of the lipid bilayer. Therefore, the study indicates the formation of an interdigitated phase with a lower order compared to the gel phase in the DPPC membrane supplemented with the IL. © 2022 American Chemical Society. All rights reserved.