We have investigated effects of substrates on self-assembly of alkanethiol molecules by means of X-ray absorption fine structure spectroscopy. Molecular orientation of hexanethiolate adsorbed on various substrates such as Cu(111), Ag(111), Ag(100), Si(111), and their oxygen-covered surfaces has been explored by C K-edge and S K-edge near-edge X-ray absorption fine structure (NEXAFS). It was revealed that the alkyl-chain orientation is not correlated to the S-C bond orientation but primarily determined by the molecular density that definitely depends on the substrate surface. Precovered oxygen overlayers do not prevent thiol molecules from being adsorbed but promote their adsorption rate. The surface oxygen atoms are removed via formation and desorption of H2O. The interface structure of a hexanethiolate monolayer on O/Cu(100) has been studied in detail by use of S K-edge surface extended X-ray absorption fine structure (SEXAFS). The SEXAFS results indicated that hexanethiol adsorption induces a lateral displacement of surface Cu atoms toward the sulfur atom of the thiolate with a concomitant breaking of the -O-Cu-O- one-dimensional chain.