Sea buckthorn has long been used as a functional food to regulate cholesterol, relieve angina, and diminish inflammation. Flavonoids are one of the main active components in sea buckthorn. We investigated the effects of sea buckthorn flavonoid (SF) treatment on two pathways that mediate inflammation, the mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) pathways, to explore the anti-inflammatory activity of SFs in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The LPS-induced over-production of nitric oxide (NO) and prostaglandin E2 (PGE2) was inhibited by SFs through a mechanism related to the modulatory effects of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) genes. Additionally, SFs downregulated the production and mRNA expression of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β. Moreover, SFs inhibited the phosphorylation of the p38 and stress-activated protein kinase/jun amino-terminal kinase (SAPK/JNK) MAPK pathways, and they reduced the nuclear translocation of NF-κB to prevent its activation by blocking the phosphorylation and degradation of inhibitor protein of NF-κB α (IκB-α). Based on these findings, SFs may exert their inhibitory effects on inflammation by regulating the release of inflammatory mediators through the MAPK and NF-κB pathways. SFs highlight the potential benefits of using functional foods with anti-inflammatory actions to combat inflammatory diseases.