High resolution spectrum of methyl lactate, a chiral α-hydroxyester, has been investigated using a molecular jet Fourier transform microwave spectrometer. High level ab initio calculations were employed to study the conformational isomerism of methyl lactate. The observed rotational spectrum confirms that the most stable conformer has an intramolecular hydrogen bond of OH⋯OC type, as predicted by the ab initio calculations. The internal rotation barrier heights of the ester methyl group and the α-carbon methyl group were calculated to be 5.4 and 14.5 kJ mol⁻¹ at the MP2/aug-cc-pVDZ level of theory for the most stable conformer. The internal rotation splittings due to the ester methyl group were observed and analyzed and the ester methyl group tunneling barrier height was determined experimentally to be 4.762 (3) kJ mol⁻¹.