Background: Canthin-6-one (CO) is an active ingredient found in Picrasma quassioides (D.Don) Benn. (PQ) that displays various biological activities including anti-inflammatory properties. Several studies reported PQ displayed neuroprotective activities, but its effects on astrocytes have not yet been investigated. Astrocytes are crucial regulators of neuroinflammatory responses under pathological conditions in the central nervous system (CNS). Proinflammatory astrocytes can induce the blood-brain barrier (BBB) breakdown, which plays a key role in the progression of neurodegenerative disorder (ND). Purpose: This study aims to investigate the anti-neuroinflammatory effects of CO in LPS-induced astrocyte activation and its underlying mechanisms in protecting the blood-brain barrier (BBB) in vitro. Methods: Mouse astrocytes (C8-D1A) were activated with lipopolysaccharide (LPS) with or without CO pretreatment. Effects of CO on astrocyte cell viability, secretions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and nitric oxide (NO) were determined. Intracellular transcriptions and translations of proinflammatory mediators, molecular signaling, [Ca] and the levels of reactive oxygen species (ROS) were evaluated by RT-PCR, western blotting, and flow cytometry, respectively. Astrocyte-conditioned medium (ACM) was further prepared for incubating endothelial monolayer (bEnd.3) grown on transwell. Endothelial disruptions were evaluated by transendothelial electrical resistance (TEER), FITC-dextran permeability and monocyte adhesion assays. Endothelial tight junctions (TJs) and molecular signaling pathways were evaluated by immunofluorescence staining and western blotting. Results: CO attenuated LPS-induced expression of astrocytic proinflammatory mediators (TNF-α, IL-1β, IL-6, NO) and inhibited deleterious molecular activities including inducible nitric oxide synthase (iNOS), p-NFκB and p-STAT3 in astrocytes. Incubation of ACM collected from CO-treated astrocytes significantly ameliorated endothelial disruptions, reduced expressions of endothelial cytokine receptors (IL-6R, gp130 (IL-6RB), TNFR and IL-1R), suppressed proinflammatory pathways, MAPKs (p-AKT, p-MEK, p-ERK, p-p38, p-JNK) and p-STAT3, restored endothelial stabilizing pathways (p-Rac 1) and upregulated beneficial endothelial nitric oxide synthase (eNOS). Conclusion: Our study demonstrates for the first time CO exhibited potent protective effects against astrocyte-mediated proinflammatory responses and associated endothelial barrier disruptions.