There are few reports on the biological activities of chaetoglobosin V(b) (Cha V(b)) (a cytochalasin alkaloid). In this study, we investigated the molecular mechanisms underlying the anti-inflammatory and antioxidant effects of Cha V(b) in the RAW264.7 cells stimulated lipopolysaccharide (LPS). LPS stimulation-induced oxidative stress (i.e. increase production of reactive oxygen species (ROS) and decreased expression of antioxidant superoxide dismutase (SOD)) was suppressed after a Cha V(b) treatment. Cha V(b) could significantly inhibit the upregulated expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene and protein induced by LPS whilst attenuating the production of pro-inflammatory cytokines TNF-alpha, IL-6 and IL-1beta. Such antioxidant and anti-inflammatory effects were achieved through the TLR4-mediated MyD88-dependent signaling pathways (via suppressing the phosphorylation of p38, ERK, JNK MAPK and translocation of the NF-kappaB p65 subunit into nucleus), and the TRIF-dependent signaling pathways (via reducing IFN-beta release without inhibiting interferon-regulated factor 3 (IRF3) and IRF7). At 25-100 muM (a concentration range with no cytotoxicity), Cha V(b) dose-dependently influenced SOD enzyme activity and phosphorylation of p38, ERK1/2 and JNK, and at 100 muM, likely exerted the greatest inhibition towards LPS-induced oxidative stress and inflammatory response via the MAPK and NF-kappaB signaling pathway. CI - Copyright (c) 2020 Elsevier Ltd. All rights reserved.