During inflammation, lower molecular weight fragments of hyaluronan accumulate, and this is known to be inflammatory and immune-stimulatory. In diseases such as inflammatory bowel disease, inflammatory cells bind to hyaluronan; however, the cellular response and molecular mechanism of hyaluronan–hyaluronan receptor interactions in mononuclear cells are not well understood. The expression of hyaluronan receptors in peripheral blood mononuclear cells (PBMC) was examined. PBMC were stimulated with lower and higher molecular weight hyaluronan (molecular weight 100–150 kDa and 2700 kDa) and the induction of proinflammatory cytokines (interleukin-6 (IL-6) and monocyte chemoattractant protein (MCP-1)) was compared by enzyme-linked immunoabsorbant assay (ELISA). Cells were coincubated with various signaling pathway inhibitors. In addition, neutralizing antibodies against CD44 and TLR4 were added and the effects on PBMC were investigated. Finally, mononuclear cells from CD44-null and toll-like receptor 4 (TLR4) mutant mice were both stimulated with lower molecular weight hyaluronan. Among the hyaluronan receptors, TLR4 and CD44 were markedly expressed on PBMC. Hyaluronan-stimulated PBMC enhanced the attachment to the extracellular matrix. Lower molecular weight hyaluronan induced IL-6 and MCP-1 production in PBMC, but high-molecular-weight hyaluronan did not induce IL-6 and MCP-1 production. An anti-CD44 antibody attenuated the induction of both IL-6 and MCP-1 in lower molecular weight hyaluronan-stimulated PBMC. In both TLR4 mutant and CD44-null mice, the induction of IL-6 by lower molecular weight hyaluronan stimulation was decreased. SB203580 completely abolished IL-6 production in both TLR4 mutant and CD44-null mononuclear cells, while PD98059 abolished IL-6 production in CD44-null mononuclear cells. Hyaluronan receptors, CD44 and TLR4, play distinct roles in cytokine induction in hyaluronan-stimulated mononuclear cells.