Background: Fisetin, a natural potent flavonoid, has various beneficial, pharmacological activities. In this study, we investigated expression changes of the fisetin regulating genes in lipopolysaccharide (LPS)-treated RAW264.7 cells and explored the role of fisetin in inflammation and autophagy. Methods and results: Microarray analysis identified 1,071 genes that were regulated by fisetin in LPS-treated RAW264.7 cells, and these genes were mainly related to the process of immune system response. Quantitative real-time polymerase chain reaction and Bio-Plex analysis indicated that fisetin decreased the expression and secretion of several inflammatory cytokines in cells administered with LPS. Western blot analysis and immunofluorescence assay showed that fisetin decreased microtubule-associated protein 1 light-chain 3B (LC3B) and lysosome-associated membrane protein 1 (LAMP1) expression in LPS-treated cells, while the autophagy inhibitor chloroquine (CQ) could partially reverse this effect. In addition, fisetin reduced the elevated expression of p-PI3K, p-AKT and p-mTOR induced by LPS in a concentration-dependent manner. Conclusions: Fisetin diminished the expression and secretion of inflammatory cytokines and facilitated autophagosome-lysosome fusion and degradation in LPS-treated RAW264.7 cells via inhibition of the PI3K/AKT/mTOR signaling pathway. Overall, the results of this study provide new clues for the anti-inflammatory mechanism of fisetin and explain the crosstalk between autophagy and inflammation to some extent.