The reactions of both thiophene and H2S on Mo2C/Al2O3 catalyst have been studied by in situ FT-IR spectroscopy. CO adsorption was used to probe the surface sites of Mo2C/Al2O3 catalyst under the interaction and reaction of thiophene and H2S. When the fresh Mo2C/Al2O3 catalyst is treated with a thiophene/H2 mixture above 473 K, hydrogenated species exhibiting IR bands in the regions 2800–3000 cm−1 are produced on the surface, indicating that thiophene reacts with the fresh carbide catalyst at relatively low temperatures. IR spectra of adsorbed CO on fresh Mo2C/Al2O3 pretreated by thiophene/H2 at different temperatures clearly reveal the gradual sulfidation of the carbide catalyst at temperatures higher than 473 K, while H2S/H2 can sulfide the Mo2C/Al2O3 catalyst surface readily at room temperature (RT). The sulfidation of the carbide surface by the reaction with thiophene or H2S maybe the major cause of the deactivation of carbide catalysts in hydrotreating reactions. The surface of the sulfided carbide catalyst can be only partially regenerated by a recarburization using CH4/H2 at 1033 K. When the catalyst is first oxidized and then recarburized, the carbide surface can be completely reproduced.