Fisetin is a naturally occurring flavonoid that possesses several pharmacological benefits including anti-inflammatory activity. However, its precise anti-inflammatory mechanism is not clear. In the present study, we found that fisetin significantly inhibited the expression of proinflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Additionally, fisetin attenuated LPS-induced mortality and abnormalities in zebrafish larvae and normalized the heart rate. Fisetin decreased the recruitment of macrophages and neutrophils to the LPS-microinjected inflammatory site in zebrafish larvae, concomitant with a significant downregulation of proinflammatory genes, such as inducible NO synthase (iNOS), cyclooxygenase-2a (COX-2a), IL-6, and TNF-α. Fisetin inhibited the nuclear localization of nuclear factor-kappa B (NF-κB), which reduced the expression of pro-inflammatory genes. Further, fisetin inactivated glycogen synthase kinase 3β (GSK-3β) via phosphorylation at Ser9, and inhibited the degradation of β-catenin, which consequently promoted the localization of β-catenin into the nucleus. The pharmacological inhibition of β-catenin with FH535 reversed the fisetin-induced anti-inflammatory activity and restored NF-κB activity, which indicated that fisetin-mediated activation of β-catenin results in the inhibition of LPS-induced NF-κB activity. In LPS-microinjected zebrafish larvae, FH535 promoted the migration of macrophages to the yolk sac and decreased resident neutrophil counts in the posterior blood island and induced high expression of iNOS and COX-2a, which was accompanied by the inhibition of fisetin-induced anti-inflammatory activity. Altogether, the current study confirmed that the dietary flavonoid, fisetin, inhibited LPS-induced inflammation and endotoxic shock through crosstalk between GSK-3β/β-catenin and the NF-κB signaling pathways.