Oxacalixarenes represent a distinctive class of macrocyclic compounds, which are closely related to the parent calixarene family, offering binding motifs characteristic of calixarenes and crown ethers. Nevertheless, they still lack extensive characterization in terms of molecular recognition properties and the subsequent practical applicability. We present here the results of binding studies of an oxacalix[4]arene carboxylate macrocycle toward a variety of organic ammonium cationic species. Our results show that the substituents attached to the guest ammonium compound largely influence the binding strengths of the host. Furthermore, we show that the characteristic binding pattern changes upon transition from the gas phase to solution in terms of the governing intermolecular interactions. We identify the key factors affecting host–guest binding efficacy and suggest rules for the important molecular structural motifs of the interacting parts of ammonium guest species and the macrocycle to facilitate sensing of ammonium cations.