NR4a1 has been demonstrated to exert a protective role in various chronic inflammatory disease. The underlying mechanism has yet to be clarified. This research aimed to explore the anti-inflammatory mechanism of NR4a1on macrophage through bioinformatics analysis and further verified by a series of experiments. In this study, The GSMs (GEO samples) that LPS(lipopolysaccharide) treated macrophage derived from wild type and NR4a1 knockout mice were extracted from GSE68167 gene expression profiles in Gene Expression Omnibus (GEO) database. DEGs (Differentially expressed genes) screened out. The gene ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes pathway) enrichment were conducted. PPI (Protein-protein interaction) network that comprised 49 nodes was mapped. The enrichment of biological process (BP) was principally and significantly revealed in signal transduction, inflammatory response and positive regulation of chemokine production. In terms of KEGG pathway, DEGs were primarily enriched in Cytokine-cytokine receptor interaction, JAK-STAT signaling pathway and cAMP signaling pathway. In the PPI network, IL10, IFNG, Fos, IL19, PDE4B NPY, Cnr1, MMP13, Rtn1, UCHL1 were selected as Hub gene. PDE4B and cAMP signaling pathway may associate with anti-inflammatory function of NR4a1. Our data showed that NR4a1 could promotes the protein level of cAMP, p-PKA and decreased p-p65, p-IκBα through down-regulating PDE4B. NR4a1-downregulated PDE4B expression notably decreased the LPS induced mRNA level of IL-6 and IL1β. This study also pointed the potential physiological processes regulated by NR4a1.Our findings supports that NR4a1 may be a considerable therapeutic target in inflammatory disease.