The triterpene glycoside glycyrrhizin is the main active compound in liquorice. It is used as a herbal medicine owing to its anticancer, antiviral and anti-inflammatory properties. Its mode of action, however, remains widely unknown. In the present study, we aimed to elucidate the molecular mechanism of glycyrrhizin in attenuating inflammatory responses in macrophages. Using microarray analysis, we found that glycyrrhizin caused a broad block in the induction of pro-inflammatory mediators induced by the TLR (Toll-like receptor) 9 agonist CpG-DNA in RAW 264.7 cells. Furthermore, we found that glycyrrhizin also strongly attenuated inflammatory responses induced by TLR3 and TLR4 ligands. The inhibition was accompanied by decreased activation not only of the NF-kappaB (nuclear factor kappaB) pathway but also of the parallel MAPK (mitogen-activated protein kinase) signalling cascade upon stimulation with TLR9 and TLR4 agonists. Further analysis of upstream events revealed that glycyrrhizin treatment decreased cellular attachment and/or uptake of CpG-DNA and strongly impaired TLR4 internalization. Moreover, we found that the anti-inflammatory effects were specific for membrane-dependent receptor-mediated stimuli, as glycyrrhizin was ineffective in blocking Tnfa (tumour necrosis factor alpha gene) induction upon stimulation with PMA, a receptor- and membrane-independent stimulus. These observations suggest that the broad anti-inflammatory activity of glycyrrhizin is mediated by the interaction with the lipid bilayer, thereby attenuating receptor-mediated signalling.