The reactive adsorption behavior of thiophene over the reduced NiZnO/Al2O3-diatomite adsorbent was characterized by in situ Fourier transform infrared (FTIR) spectroscopy. X-ray diffraction (XRD) technology was used to investigate the phase change of sulfur species in the reactive adsorption desulfurization (RADS) process. The results indicated that S-M bonding of thiophene on the metallic Ni sites was first decomposed to form Ni3S2 while formed C-4 olefins were further saturated by hydrogen to form butane which was released back into the process stream, followed by the sulfur transfer from Ni3S2 to ZnO to form ZnS in the presence of hydrogen, and then the new formed Ni sites could participate in the adsorption of thiophene once again. The muticycle fixed-bed tests showed a good prospect for adsorption desulfurization over the NiZnO/Al2O3-diatomite adsorbents. Thermogravimetric and differential thermal analysis (TG-DTA) together with XRD was used to reveal the regeneration mechanism. The XRD results indicated that the formation of NiSO4 species led to an increase of the amount and the strength of Lewis acid sites in the regenerated adsorbents and, thus, temporarily improved the removal performance of the adsorbent for thiophene.