•The anti-inflammatory effect of trilobatin and its mechanism were investigated.•Trilobatin treatment reduced the LPS-stimulated TNF-α, IL-1β, and IL-6 expression.•Trilobatin treatment inhibited the IκB degradation and NF-κB p65 phosphorylation.We investigated the anti-inflammatory effect of trilobatin, the flavonoid isolated from the leaves of Lithocarpus polystachyus Rehd, as well as the underlying molecular mechanisms. Treatment with trilobatin (0.005–5 μM) dose-dependently inhibited the lipopolysaccharide (LPS)-induced mRNA expression and secretion of pro-inflammatory cytokines, including tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) and interleukin-6 (IL-6), in RAW 264.7 macrophages. However, no further inhibition was detected when the concentration of trilobatin was increased to 50 μM. Western blot analysis confirmed that the mechanism of the anti-inflammatory effect was correlated with the inhibition of LPS-induced inhibitor of nuclear factor-kappa B α (IκBα) degradation and nuclear factor-kappa B (NF-κB) p65 phosphorylation. In addition, trilobatin also showed a significant inhibition of LPS-induced TNFα and IL-6 at both the mRNA and protein levels in a mouse model. Our results suggest that trilobatin potentially inhibits the LPS-induced inflammatory response by suppressing the NF-κB signaling pathway.