Bacterial populations rely on quorum sensing (QS) to coordinate their behaviors and are often challenged by the fluctuation in oxygen concentrations in their habitats. Oxygen is a crucial factor that affects bacterial metabolism in multiple ways. However, little is known about whether and how oxygen availability affects QS activities. To fill this gap, we used the facultative anaerobe Novosphingobium pentaromativorans US6-1 as a model system, and observed that the QS signal acyl homoserine-lactones (AHLs) were produced only in anoxic environments, such as biofilm, or liquid medium that initially contained less than 2 mg/L dissolved oxygen, but not in highly oxic environments. Comparative transcriptome analysis revealed that oxygen availability significantly affected the physiological activities in US6-1, including fatty acid metabolism, oxidative phosphorylation, citrate cycle, QS activities, and flagellar assembly. The absence of AHLs in the oxic culture was not due to degradation, but to the very low expression of the AHL synthase gene novI. High concentration of NADH during the middle log phase under static cultivation may be a trigger for AHL synthesis. This is the first report that production of AHLs is coupled with anoxic metabolism in a facultative anaerobe, which extends our knowledge on factors affecting bacterial QS occurrence. KEY POINTS: • AHL production is anoxic cultivation related. • Oxygen availability affects AHL synthesis by influencing novI expression. • Oxygen availability changes many metabolism activities including NADH production.