Luteolin, a natural flavonoid exists in various medicinal plants, has strong anti-inflammatory effect. However, anti-inflammatory mechanism of luteolin has not been fully explored. Hence, we systematically investigated druggability and anti-inflammatory mechanism of luteolin based on network pharmacology and in vitro experiments. The absorption, distribution, metabolism and excretion of luteolin were evaluated by TCMSP server. Targets associated with luteolin and inflammation were collected from public databases, and the overlapping targets between luteolin and inflammation were analyzed by Draw Venn diagram. Then the protein-protein interaction network of luteolin against inflammation was constructed. Further, gene function and pathway enrichment analysis were performed. Finally, in vitro experiments were carried out to estimate the accuracy of predicted target genes. ADME results indicated that luteolin has great potential to be developed into a drug. 226 overlapping targets were screened by matching 280 targets of luteolin with 9015 targets of inflammation. 9 core targets of luteolin against inflammation were identified, including MMP9, MAPK1, HSP90AA1, CASP3, ALB, EGFR, SRC, HRAS and ESR1. Gene function were mainly involved in metabolism, energy pathways and signal transduction. Metabolic pathways, pathways in cancer, PI3K-AKT signaling pathway, Ras signaling pathway and so on might be the critical pathways of luteolin against inflammation. RT-qPCR and ELISA results indicated that luteolin decreased the expression of most of core genes at protein and mRNA levels (MMP9, MAPK1, HSP90AA1, EGFR, SRC and HRAS). Luteolin is expounded to have great potential to be developed into a drug and target various genes and pathways to perform anti-inflammatory effect. Copyright © 2020. Published by Elsevier B.V.