Punicalagin (2,3,hexahydroxydiphenoyl-gallagyl-D-glucose and referred to as PUN) is a bioactive ellagitannin isolated from pomegranate, which is widely used for the treatment of inflammatory bowel disease (IBD), diarrhea, and ulcers in Chinese traditional medicine. In this study, we detected the anti-inflammation potentials of PUN in lipopolysaccharide (LPS)-induced macrophages and tried to uncover the underlying mechanism. Results demonstrated that PUN (25, 50, or 100 muM) treatment could significantly decrease the LPS-induced production of nitric oxide), prostaglandin E2 (PGE2), interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha in RAW264.7 cells. Molecular research showed that PUN inhibited the activation of upstream mediator nuclear factor-kappaB by suppressing the phosphorylation of IkappaBalpha and p65. Results also indicated that PUN could suppress the phosphorylation of mitogen-activated protein kinase including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase. In conclusion, we observed that PUN could inhibit LPS-induced inflammation, and it may be a potential choice for the treatment of inflammation diseases.