Amlexanox, an anti-inflammatory agent, is widely used for treating aphthous ulcers. Recently, amlexanox has received considerable attention because of its efficacy in mitigating metabolic inflammation via directly suppressing IKKε/TBK1. However, because the knockdown of IKKε/TBK1 has no anti-inflammatory effect on lipopolysaccharide (LPS)-primed RAW264.7 cells, the mechanism of amlexanox against classical inflammation is independent of IKKε/TBK1. In this study, we aim to examine the effects of amlexanox on LPS-treated macrophages and in a mouse model of endotoxemia. We found that amlexanox significantly inhibited the production of pro-inflammatory mediators, both in vitro and in vivo, while increased interleukin-10 level in LPS-activated macrophages. Mechanistically, amlexanox down-regulated nuclear factor κB and extracellular signal-regulated kinase/activator protein-1 signaling by elevating intracellular 3',5'-cyclic adenosine monophosphate (cAMP) level and subsequently activating protein kinase A. Molecular docking along with fluorescence polarization and enzyme inhibition assays revealed that amlexanox bound directly to phosphodiesterase (PDE) 4B to inhibit its activity. The anti-inflammatory effects of amlexanox could be abolished by the application of cAMP antagonist or PDE4B siRNA. In addition to PDE4B, the activities of PDE1C, 3A, and 3B were directly inhibited by amlexanox. Our results provide mechanistic insight into the clinical utility of amlexanox for the treatment of inflammatory disorders and might contribute to extending the clinical indications of amlexanox.