The importance of di-nitrogen (N2) production via aerobic pathways has been verified in a significant number of pure microbial strains. However, to date there are no reports confirming this in situ. In this study, we extracted micro-biota from three typical upland soils with variable properties and incubated them under aerobic and anaerobic conditions, with nitrate, to investigate whether N2 production occurred via aerobic pathways and the relative importance of it when compared with the N2 production via anaerobic pathways. Our results showed that N2 can be produced in soil extracts under aerobic conditions, and that the N2 produced via aerobic pathways equated to 29–51% of that produced via anaerobic pathways. Thus N2 production via aerobic pathways may play a significant role in soil nitrogen (N) cycling. Our results demonstrate that an O2 deficit may not to be a requirement for converting reactive N back into inert N2, and consequently this process may be more widespread in upland soils than currently thought.