Nitric oxide (NO) is an important mediator of inflammation response and the production of NO has been linked to a variety of diseases, including tumors, inflammation and central nervous system diseases. In macrophages, a high level of NO is generated by iNOS during inflammatory responses triggered by cytokines or pathogens. Autophagy, a cellular bulk degradation process via lysosome, has been implicated in many disease conditions including inflammation. In this study, we have reported the previously unknown role of autophagy in regulating iNOS levels in macrophages, both under basal and Lipopolysaccharides (LPS)-induced conditions. Our data showed that iNOS levels accumulated upon autophagy inhibition and decreased upon autophagy induction. iNOS interacted and co-localized with autophagy receptor p62/SQSTM1, especially under LPS-stimulated condition in macrophages. Moreover, the immunostaining data revealed that iNOS also co-localizes with the autophagosome marker LC3 and lysosome marker LAMP1, especially under lysosomal inhibition conditions, indicating iNOS is an autophagy substrate. Finally, we showed that autophagy negatively regulated the generation of NO in macrophages, which is consistent with the changes of iNOS levels. Collectively, our study revealed a previously unknown mechanism by which autophagy regulates iNOS levels to modulate NO production during inflammation.