In the present work, we report the first rotational study of N-acetylgalactosamine, a cancer-associated sugar derivative, by means of high-resolution rotational spectroscopy. Two different conformers have been conclusively characterized using broadband Fourier transform microwave spectroscopy coupled with a laser ablation vaporization system. Additionally, we performed a comprehensive analysis of the intramolecular interactions that govern these structures, which allowed us to both characterize the existence of intramolecular hydrogen bond networks that drive the intrinsic conformation panorama of N-acetylgalactosamine and further rationalize the biological role of this aminosugar derivative as part of the Tn antigen.