The purpose of this study was to evaluate the effects of polydatin (PD) on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions at protein and transcriptional levels, as well as the production of prostaglandin E2 (PGE2) and nitric oxide (NO) in lipopolysaccharide (LPS)-induced macrophage RAW 264.7 cells. To elucidate the underlying mechanism responsible for these symptoms, we investigated the phosphorylation of mitogen-activated protein kinase (MAPK) pathway and nuclear factor-κB (NF-κB) expression. NO was analyzed with the Griess method. PGE2 was measured by enzyme-linked immunosorbent assay (ELISA). iNOS and COX-2 messenger RNA (mRNA) were identified by qPCR assay. iNOS, COX-2, NF-κB, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 protein expressions were detected with Western blot. The results revealed that PD effectively inhibited NO and PGE2 production, and it is not surprising that PD reduced iNOS and COX-2 expression at protein and transcriptional levels. Additionally, PD significantly ameliorated the activation of NF-κB and the phosphorylation of MAPKs in LPS-induced RAW 264.7 macrophages. These findings suggested that PD exerted potent anti-inflammatory activity in macrophages.