Prostaglandin E₂ (PGE₂) is one of the key mediators of inflammation in affected joints of rheumatoid arthritis (RA). Intra-articular injection of high molecular weight hyaluronan (HA) into RA knee joints relieves arthritic pain. Although HA has been shown to inhibit PGE₂ production in cytokine-stimulated synovial fibroblasts, it remains unclear how HA suppresses PGE₂ production in activated cells. Furthermore, HA effect on macrophages has rarely been investigated in spite of their contribution to RA joint pathology. This study was aimed to investigate the inhibitory mechanism of HA on lipopolysaccharide (LPS)-stimulated PGE₂ production in U937 human macrophages. Stimulation of U937 macrophages with LPS enhanced PGE₂ production in association with increased protein levels of cyclooxygenase-2 (COX-2). Pretreatment with HA of 2,700 kDa resulted in suppression of the LPS-mediated induction of COX-2, leading to a decrease in PGE₂ production. Likewise, the LPS-stimulated PGE₂ production was inhibited by the pretreatment with a specific COX2 inhibitor, NS-398, or a specific inhibitor of nuclear factor (NF)-κB, BAY11-7085. HA also decreased the degree of phosphorylation and nuclear translocation of NF-κB enhanced by LPS. Fluorescence cytochemistry demonstrated that HA bound to CD44, the principal HA receptor, on U937 macrophages. Anti-CD44 antibody reversed the inhibitory effects of HA on the LPS-mediated increase in PGE₂ production, COX-2 induction, and activation of NF-κB. These results indicate that HA suppresses the LPS-stimulated PGE₂ production via CD44 through down-regulation of NF-κB. Administration of HA into RA joints may decrease PGE₂ production by activated macrophages, which could result in improvement of arthritic pain.