A detailed structural characterization of organo-clays is a key in understanding their properties. In this work, mono-, di-, tri-, and tetra-butylammonium (nBA; n = 1–4) cations intercalated in the layered clay mineral montmorillonite (Mnt) have been studied for the first time by combining a theoretical approach based on density functional theory (DFT) and infrared spectroscopy. The DFT calculations revealed the detailed structure and position of nBA cations in the interlayer space. A relation between the basal spacing (d001 parameter) and the cation size and structure was found, and explained with respect to the structure, composition, and size of the organic cations. Hydrogen bonds between –NHx/–CH3/–CH2 groups of the nBA cations and oxygen atoms of the basal planes of the Mnt layers were found to be an important factor for the arrangement and energetic stabilization of cations in the interlayer space. The N–H⋯O hydrogen bonds are stronger than C–H⋯O hydrogen bonds and the stabilization decreases with decreased number of bands. Analysis of DFT-calculated vibrational modes helped in understanding a problematic region of the experimental infrared spectra (4000–3000 cm–1), in which assignment of all vibrational modes unambiguously was not possible because of a significant overlap of broad bands.