To achieve efficient low-content NO oxidation at room temperature is a hot but challenging topic in heterogeneous catalysis, and MnO2-based oxide catalysts have recently drawn so much attention owing to the potential activity. However, the activity origin of MnO2 and the critical rate-limiting factor are vague, impeding further catalyst optimization. Herein, by combining the first-principles calculations and microkinetic analyses, we systematically investigate the low-content NO oxidation process catalyzed by MnO2. On MnO2(110) exposed by two kinds of active sites (Mn5c and the lattice Obri), the favorite pathway contributing to NO oxidation is figured out by examining the complicated reaction network. This reveals that the Mars-van Krevelen mechanism with the lattice Obri involved is preferred rather than the Langmuir-Hinshelwood one occurring at the Mn5c sites alone. First, NO adsorbs at Mn5c (as NO∗) and is oxidized by the lattice Obri, forming NO2# (# denotes the Obri vacancy, Ovac) that can desorb with an Ovac left. Second, O2 can adsorb at Ovac (O2#) and react with NO∗ into an intermediate ONOO, which can break its O-O bond and release NO2. It is also found that Mn5c can exclusively adsorb NO and guarantee the coverage of NO∗ even for the low-content NO(g) and the lattice Obri is very reactive and provides oxidative species, showing a synergetic catalytic role accounting for high activity of MnO2 for low-content NO oxidation at room temperature. Quantitatively, the adsorption energies of NO at Mn5c (Eads(NO@Mn5c)) and O2 at Ovac (Eads(O2@Ovac)) can serve as the important activity descriptors and increasing either of them could improve the activity of MnO2. In addition, our microkinetic results show that the NO# (NO adsorbed at the Ovac) would be a key poisoning species and deactivate MnO2 owing to its stronger adsorption in comparison with O2, whereas the nitrate/nitrite species could not cause the blockage of active sites as expected. This work could provide a significant insight into low-content NO oxidation at room temperature catalyzed by MnO2. © 2018 American Chemical Society.