The influence of an acetamido group in directing the preferred choice of hydration sites in glucosamine and a consequent extension of the working rules governing regioselective hydration and conformational choice, have been revealed through comparisons between the conformations and structures of “free” and multiply hydrated phenyl N‐acetyl‐β‐D‐glucosamine (βpGlcNAc) and phenyl β‐D‐glucopyranoside (βpGlc), isolated in the gas phase at low temperatures. The structures have been assigned through infrared ion depletion spectroscopy conducted in a supersonic jet expansion, coupled with computational methods. The acetamido motif provides a hydration focus that overwhelms the directing role of the hydroxymethyl group; in multiply hydrated βpGlcNAc the water molecules are all located around the acetamido motif, on the “axial” faces of the pyranose ring rather than around its edge, despite the equatorial disposition of all the hydrophilic groups in the ring. The striking and unprecedented role of the C‐2 acetamido group in controlling hydration structures may, in part, explain the differing and widespread roles of GlcNAc, and perhaps GalNAc, in nature. Selective hydration: The acetamido motif provides a hydration focus that locates bound water molecules on the “axial” faces of the pyranose ring—a striking and unprecedented role that may, in part, explain the differing and widespread role of GlcNAc in nature.