Abstract The conformational preferences of o -vanillin have been investigated in a supersonic jet expansion using Fourier transform microwave (FT-MW) spectroscopy. Three molecular conformations were derived from the rotational spectrum. The two most stable structures are characterized by a moderate O H···O C hydrogen bond between the aldehyde and the hydroxyl groups, with the methoxy side chain either in plane (global minimum a- cis–trans ) or out of plane (a- cis-gauche ) with respect to the aromatic ring. In the third conformer the aldehyde group is rotated by ca. 180°, forming a O H···O hydrogen bond between the methoxy and hydroxyl groups (s- trans–trans ). Rotational parameters and relative populations are provided for the three conformations, which are compared with the results of ab initio (MP2) and density-functional (B3LYP, M05–2X) theoretical predictions.