V2O5–WO3, supported on a TiO2 catalyst, was tested for n-butane oxidative dehydrogenation with NO/O2. The effect of the physical oxygen mobility and nature in the V2O5–WO3/TiO2 catalyst on the applicative catalytic performance was investigated. The influence of the O2/NO species on the chemical reaction products/Mars–van Krevelen (MvK) cycle was examined by performing online time studies with the removal of an oxidant (O2/NO) in the feed. A high rate of selectivity towards butadiene was obtained when NO was used. No trace concentration of NO was observed at commonly higher temperatures. N2O or NO2 was not present in the effluent gas compounds, and the mechanism was analyzed. In the presence of both O2/NO, a high synthesis yield of diene was determined at lower reactant conversions. The former increased up to 40% n-butane conversion, and decreased with a further increase in n-butane conversion. X-ray photoelectron spectroscopy (XPS) showed that the re-oxidation with NO restored the lattice oxygen (O2−) in the catalyst, along with the surface oxygen species. The catalyst reduced at 350 °C showed the presence of the superficial lattice oxygen species (O−/O2−) generated from the lattice defects, which also involved the interstitial oxygen species. The catalyst reduced at 450 °C showed the presence of the O2− radical anion with the co-existence of O2−. The product distribution showed that the butane dehydrogenation to butene and butadiene occurred, and that the insertion of oxygen into the catalyst lattice favored butadiene production.