To investigate the mechanism of fuel NO formation, an experimental study using a flow reactor and a numerical simulation with a detailed kinetic model were performed. Pyridine was used as a model compound of fuel nitrogen, and the effects of oxygen and H2O concentrations on reaction mechanism was investigated. The results showed that oxygen concentration had little effect on the reaction mechanism, while NO formation was inhibited with an increase in H2O concentration. In this reaction mechanism, NO is mainly formed by the NCO-O reaction, which is an important reaction for NO formation. At high H2O concentration, many O radicals are consumed by the O+H2O reaction. Therefore, the formation of NO is reduced with an increase in H2O concentration.