Aureusidin, a naturally-occurring flavonoid, is found in various plants of Cyperaceae such as Heleocharis dulcis (Burm. f.) Trin., but its pharmacological effect and active mechanism are rarely reported. This study aimed to investigate the anti-inflammatory effect and action mechanism of Aureusidin in LPS-induced mouse macrophage RAW264.7 cells. The results suggested that lipopolysaccharide (LPS)-induced nitric oxide (NO), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) production were obviously inhibited by Aureusidin. Moreover, Aureusidin also significantly decreased the mRNA expression of various inflammatory factors in LPS-stimulated RAW264.7 cells. Furthermore, mechanistic studies showed that Aureusidin significantly inhibited nuclear transfer of nuclear factor-κB (NF-κB), while increasing the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) as well as expression of Nrf2 target genes such as heme oxygenase (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), but the addition of the HO-1 inhibitor Sn-protoporphyrin (Snpp) significantly abolished the anti-inflammatory effect of Aureusidin in LPS-stimulated RAW264.7 cells, confirming the view that HO-1 was involved in the anti-inflammatory effect. In addition, Aureusidin increased the levels of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) phosphorylation in RAW264.7 cells. Antioxidant N-acetylcysteine (NAC) or three MAPK inhibitors blocked the nuclear translocation of Nrf2 and HO-1 expression induced by Aureusidin, indicating that Aureusidin activated the Nrf2/HO-1 signaling pathway through ROS and MAPKs pathways. At the same time, co-treatment with the NAC blocked the phosphorylation of MAPKs. Results from molecular docking indicated that Aureusidin inhibited the NF-κB pathway by covalently binding to NF-κB. Thus, Aureusidin exerted the anti-inflammatory activity through blocking the NF-κB signaling pathways and activating the MAPKs and Nrf2/HO-1 signaling pathways. Based on the above results, Aureusidin may be an attractive therapeutic candidate for the inflammation-related diseases.