MnO2-based catalysts exhibit remarkable activities in a room-temperature NO oxidation reaction (RT-NOR) but are challenged by water-induced deactivation. To improve the water-resistance driven by molecular-level mechanistic understanding, we herein report a systematic first-principles study and microkinetic simulation on elucidating the water-modulated RT-NOR mechanism on the notable β-MnO2 catalyst. Results show that RT-NOR preferentially follows the Mars-van Krevelen mechanism on β-MnO2(110) and that the coupling of lattice O (Olat) with NO sitting on the surface Mncus site is the rate-limiting step. The presence of H2O results in a competitive effect against NO adsorbing on Mncus and inhibits NO2 formation. More crucially, the H2O-dissociated H species eventually accumulate on Olat and are difficult to remove, which seriously poison the catalyst, although the Mncus-anchored OH intermediate can oxidize NO to NO2 via the nitrite/nitrate route. Weakening the H adsorption on Olat, as well as the H2O adsorption on Mncus, is thus proposed as conductive to alleviating MnO2 deactivation. More essentially, we find that the vacancy formation energy of Olat, Ef(Ovac), can serve as a unified descriptor for balancing H2O-resistance and catalytic activity; the excessively low Ef(Ovac) is the intrinsic origin of MnO2 susceptible to deactivation by H2O. By doping engineering guided by such a descriptor, we identify that the Cr-, Nb-, Ru-, or Rh-substituted doping can properly increase the Ef(Ovac) of MnO2 and weaken the H2O-induced deactivation in RT-NOR. These results may provide a theoretical basis on optimizing MnO2-based catalysts with improved H2O-resistance.