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Adenosine deaminase (ADA) is an enzyme distributed in a wide variety of organisms that cleaves adenosine into inosine. Since inosine plays an important role in nitrogen metabolism, ADA may have a critical function in the regulation of fatty acid synthesis. However, the role of ADA in oleaginous fungi has not been reported so far. Therefore, in this study, we identified one <i>ada</i> gene encoding ADA (with ID scaffold0027.9) in the high lipid-producing fungus, <i>Mucor circinelloides</i> WJ11, and investigated its role in cell growth, lipid production, and nitrogen metabolism by overexpressing and knockout of this gene. The results showed that knockout of the <i>ada</i> altered the efficiency of nitrogen consumption, which led to a 20% increment in the lipid content (25% of cell dry weight) of the engineered strain, while overexpression of the <i>ada</i> showed no significant differences compared with the control strain at the final growth stage; however, interestingly, it increased lipid accumulation at the early growth stage. Additionally, transcriptional analysis was conducted by RT-qPCR and our findings indicated that the deletion of <i>ada</i> activated the committed steps of lipid biosynthesis involved in acetyl-CoA carboxylase (<i>acc1</i> gene), cytosolic malic acid enzyme (<i>cme1</i> gene), and fatty acid synthases (<i>fas1</i> gene), while it suppressed the expression of AMP-activated protein kinase (<i>ampk α1</i> and <i>ampk β</i> genes), which plays a role in lipolysis, whereas the <i>ada</i>-overexpressed strain displayed reverse trends. Conclusively, this work unraveled a novel role of ADA in governing lipid biosynthesis and nitrogen metabolism in the oleaginous fungus, <i>M. circinelloides</i>.