In this study, we examined the conformation and intermolecular interactions of benzo-12-crown-4 (B12C4) complexes with NH4 +, CH3NH3 + (MeNH3 +), CH3CH2NH3 + (EtNH3 +), and CH3CH2CH2NH3 + (PrNH3 +) using cold gas-phase spectroscopy. All of the B12C4 complexes showed sharp vibronic features in the UV photodissociation spectra, and the position of the 0-0 band was close to that of the B12C4 complex with an isotropic K+ guest. This result suggests that the conformation of B12C4 is maintained despite oriented interactions with ammonium guests via anisotropic N–H···O interactions. Further, we measured the IR–UV double-resonance spectra of these complexes in the NH stretching region. In the IR–UV spectra of the EtNH3 + and PrNH3 + complexes, two distinct IR fingerprints were observed depending on the UV probe wavelength selected, indicating the existence of another (second) conformer for these complexes. Quantum chemical calculations clarified that the second conformer of the EtNH3 + and PrNH3 + complexes was partially stabilized by the C–H···π hydrogen bond. The conformation of B12C4 complexes with ammonium ions is strongly affected by the interaction between the alkyl chain of the ion guest and the benzene ring of the B12C4 host, although the main intermolecular interaction occurs between the NH3 + group and crown cavity through the N–H···O hydrogen bonds.