Berberine, an isoquinoline alkaloid in many medicinal herbs, has been found to possess broad pharmacological activities. A series of novel C-9-O-substituted-berberine derivatives have been synthesized and their anti-inflammatory effects evaluated both in vitro and in vivo. Compared to berberine, the new synthetic berberine derivatives 3i and 5e exhibit significantly improved inhibitory activities against the release of NO, TNF-α and IL-6. Furthermore, derivatives 3i and 5e were found to inhibit more effectively the migration of neutrophils and primitive macrophage in transgenic zebrafish larvae with injury-provoked inflammation. Pre-treatment with derivatives 3i or 5e could lead to a concentration-dependent decrease in nuclear factor-κB (NF-κB) p65 and NF-κB inhibitor α (IκBα) phosphorylation and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-induced RAW264.7 cells, which suggested that the anti-inflammatory activities of berberine derivatives 3i and 5e are related to their suppression of the NF-κB signal pathway.