The effect of sulfur hydrogen bonding on the conformational equilibrium of methyl 3-mercaptopropionate was investigated using microwave spectroscopy in a supersonic jet expansion. The two most stable conformers (I and II) were assigned in the rotational spectra, and complex splitting patterns owing to the methyl internal rotation and SH tunneling motion were resolved and analyzed in detail. For both conformers, the experimental torsional barriers for the methyl top are similar and about 5.1 kJ mol(-1), revealing that their geometrical differences do not affect the methyl internal rotation. The experimentally derived rotational and centrifugal distortion constants, along with the methyl internal rotation barriers, are discussed and compared with results from density functional theory and ab initio calculations. Quantum theory of atoms in molecules, noncovalent interactions, and natural bond orbital analyses show that the global minimum geometry (I), which has the thiol hydrogen oriented toward the carbonyl of the ester, is stabilized by an SH center dot center dot center dot O=C hydrogen bond. The presence of a hydrogen bond is confirmed by the derivation of an accurate experimental geometry that reveals a hydrogen bond distance and S-H-O angle of 2.515(4) angstrom and 117.4(1)degrees, respectively. These results are key benchmarks to expand the current knowledge of sulfur hydrogen bonds and the relationship between internal motions and conformational preferences in esters.