Crystalline samples of D-xylose have been vaporized by laser ablation and probed in the gas phase using Fourier transform microwave spectroscopy. The rotational spectrum revealed the existence of two α-D-xylopyranose conformers stabilized by the anomeric effect and cooperative hydrogen bond networks. The experiment spectroscopically tracked fine structural changes upon clockwise and counterclockwise arrangements of the OH groups in the observed conformers. The five monosubstituted ¹³C species of the most abundant conformer cc-α-⁴C₁ have also been observed in their natural abundance, and its structure has been derived. This work demonstrates the pivotal role that the intramolecular hydrogen-bonding network plays in the conformational behavior of free monosaccharides.