We report the bonding structure of the selenophene molecules adsorbed on the Si(100)-2 × 1 surface at 300 K, and its evolution upon annealing investigated by adopting core-level photoemission spectroscopy, near-edge X-ray absorption fine structure (NEXAFS), and ab initio calculations. The Si 2p, C 1s, Se 3d core-level spectra measured at two temperatures, 300 and 350 K, are consistently interpreted in terms of the two major structures suggested by theory, a twisted (T) 2,5-dihydroselenophene (T-DHS) and a T-deseleniumization where the selenium atom is dissociated from the selenophene ring. We find a significantly enhanced deseleniumization of selenophene molecules by mild thermal annealing indicating that these two equally abundant structures at 300 K become a single uniform phase of the T-deseleniumization structures at 350 K by overcoming a relatively low dissociation energy barrier between the two structures. In addition, we obtain an average tilt angle of a selenophene ring at 300 K from our NEXAFS spectra α53 ± 3°, which represents an ensemble average of the tilt angles, α = 15° of the T-DHS and 75° of the T-deseleniumization.