In situ scanning tunneling microscopy (STM) observation was performed to study the self-assembled monolayers of 3-mercaptopropionic acid (MPA) on Au(111) in aqueous perchloric acid solution. It was seen that the MPA molecules formed a monolayer composed of molecularly ordered domains. High-resolution STM imaging revealed that each individual MPA molecule appeared as two spots, corresponding to the carboxylic functional group of the MPA molecule, and the ordered MPA monolayer was explained by a rhombic (3 x 3) structure. The packing arrangement of the MPA monolayer is close to that of the (root3 x root3)R30 degrees structure, but possesses slightly different positions of molecules, indicating that three adjacent MPA molecules in triangular positions are snuggled up due to intermolecular hydrogen bonding. The reductive desorption reaction in alkaline solution was used to estimate the surface concentration of MPA molecules on Au(111), which is consistent with the (3 x 3) structure obtained from the STM observation. The in situ STM imaging successfully visualized the interfacial structure of MPA monolayers self-assembled on Au(111) at a molecular scale.