Pre-exposure to lipopolysaccharide (LPS) leads to hyposensitivity to secondary LPS stimulation, known as endotoxin tolerance. The role of macrophage scavenger receptor-A (SR-A) in endotoxin tolerance is unknown. In this study, LPS was shown to induce SR-A expression in the mouse macrophage cell line, RAW264.7, in dose- and time-dependent manners, which correlated with inflammatory cytokine suppression in RAW264.7 on secondary LPS stimulation. Over-expression of SR-A in RAW264.7 suppressed tumour necrosis factor (TNF)-alpha release and nuclear factor (NF)-kappa B activation, demonstrating the involvement of SR-A in endotoxin tolerance. LPS-pre-treated RAW264.7 cells could bind and internalize more fluorescein isothiocyanate (FITC)-LPS than untreated cells and both the SR-A ligand, fucoidan, and anti-SR-A 2F8 antibodies completely suppressed LPS-induced binding and internalization of FITC-LPS by RAW264.7. LPS-induced SR-A expression on RAW264.7 was completely suppressed by the p38-specific inhibitor, SB203580, but not by inhibition of toll-like receptor 4 (TLR4) signalling with MTS510, demonstrating that p38- but not TLR4-dependent up-regulation of SR-A was involved in endotoxin tolerance through binding and internalization of LPS.