Piperine exhibits various biological activities, including anti-inflammatory properties, but its anti-inflammatory mechanisms has not yet been completely elucidated. In this study, the anti-inflammatory mechanisms of piperine were elucidated at the transcriptome level based on the inflammation model of immortalized bone marrow-derived macrophages (iBMDM) of mice and transcriptome sequencing analysis. The research results revealed that piperine had no toxic effect on iBMDM within the tested concentrations, and piperine (10 μmol/L) significantly inhibited the release of inflammatory factor IL-1β (P<0.05). Transcriptome sequencing results indicate that piperine significantly up-regulated the expression levels of 35 genes and down-regulated the expression levels of 11 genes. Gene Ontology (GO) function enrichment analysis indicated that the differentially expressed genes (DEGs) in response to the piperine treatment were predominantly enriched in biological processes such as bioregulation and metabolism, along with molecular functions including cell binding, catalysis, transcriptional regulation. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis showed that DEGs were significantly enriched in inflammationrelated pathways, such as the NOD-like receptor signaling pathways and calcium signaling pathways. Reactome enrichment analysis showed the significant enrichment of DEGs in inflammatory response-related pathways, including P2X7-mediated transmembrane channels, NLRP3 inflammasomes, and P2X7-mediated intracellular potassium efflux. The results of this study elaborate the mechanisms underlying piperine's multi-target multi-pathway anti-inflammatory effect at the transcriptome level, providing a basis for further more comprehensive understanding of the molecular mechanisms underlying piperine's anti-inflammatory effect.