The conformation of benzo-18-crown-6-ether (B18C6) and its encapsulation of water molecules in a supersonic beam are investigated by laser induced fluorescence (LIF), UV–UV hole-burning, IR–UV double resonance (IR–UV DR), and resonance-enhanced multiphoton ionization (REMPI) spectroscopy with the aid of density functional theory (DFT) calculations at the B3LYP/6-31+G* level. At least four B18C6 conformers and nine B18C6–(H2O)n (n = 1–4) clusters are identified in the supersonic beam. IR–UV DR spectra in the CH stretching region suggest that the four B18C6 conformers have different conformations from each other. In contrast, most of the nine B18C6–(H2O)n clusters have a very similar B18C6 conformation. IR–UV DR spectra in the OH stretching region provide quite clear pictures of the hydration networks formed on B18C6. In all four B18C6–(H2O)1 isomers, the water molecule is H-bonded to the two O atoms adjacent to the benzene ring in “bidentate” and “bifurcated” manners. One of the four B18C6–(H2O)1 isomers exhibits a large population, and further hydration networks are preferentially grown on this specific isomer.