Rotational spectra of 3-mercaptopropionic acid (3-mpa) were measured from 6 to 18 GHz using chirped pulse and cavity-based Fourier transform microwave (FTMW) spectroscopy. Theoretical calculations using the B3LYP-D3BJ and MP2 methods with the aug-cc-pVTZ basis set show the presence of seven energy minima of 3-mpa below 8.4 kJ mol−1. Comparison of calculated rotational and centrifugal distortion constants with experimental data led to the unequivocal assignment of the global energy minimum geometry. This conformer has the mercaptan hydrogen directly oriented towards the carbonyl oxygen of the carboxylic acid moiety suggesting the presence of a six–membered ring SH…OC intramolecular hydrogen bond. Quantum theory of atoms in molecules, noncovalent interactions, and natural bond orbital analyses confirm the stability of the sulfur hydrogen bond which plays a key role in the conformational preferences of 3-mpa.