Experiments and calculations are combined to study the Nb substitution effects on the hydrodesulfurization (HDS) performance of 4,6-DMDBT catalyzed using the Ni-Mo bimetal hydrotreating catalysts. In the experimental parts, a series of Nb post-modified NiMo/Al2O3 HDS catalysts are prepared and characterized. Then, the HDS performance of the 4,6-DMDBT is assessed using this Nb-NiMo/Al2O3. In the calculations part, the Ni-Mo-S active nanocluster model with different Nb substitution degrees for the Mo-edge and Ni-edge are established. The Nb stability on the Ni-Mo-S active sites and the Nb effects on the properties of the active sites are analyzed. Meanwhile, the key elementary reactions of the direct hydrodesulfurization (DDS) route and the hydrogenation reaction (HYD) route on the Ni-Mo-S active sites are calculated. The experiments show that the introduction of Nb leads to the agglomeration of the NiMo phase in the oxidation state. Also, the introduction of Nb will increase the length and stacking number of the NiMoS phase. Partial Nb species will convert to Nb2+ during the sulfuration process. A moderate Nb introduction will enhance the HDS conversion rate, the DDS selectivity and the TOF of the 4,6-DMDBT, whereas these factors will decrease when excess Nb is introduced. The DFT calculations show that the Nb could be stable on the S-edge and Mo-edge with various substitution rates. The Nb will spatially expand the molecular orbital on the active sites, and it has a stronger interaction with the H2S, active hydrogen, 4,6-DMDBT and its DDS intermediate in general. The partial Nb substitution enhances the C-S bond cleavage. The full Nb substitution hinders the C-S bond cleavage, but it favors the hydrogen saturation of the S-4Nb. The calculations could explain the experiments in which the DDS selectivity of the 4,6-DMDBT increases with the Nb introduction at first and then decreases as the Nb introduction continues to increase.