Violaxanthin is a major carotenoid of microalgae Chlorella ellipsoidea and is also found in dark-green leafy vegetables, such as spinach. In this study, the anti-inflammatory effect of violaxanthin isolated from C. ellipsoidea was examined using lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse macrophage cells. In addition, the anti-inflammatory activity and mechanism of action of purified violaxanthin was assessed using various assays, such as quantitative real-time polymerase chain reaction (PCR), Western blotting, and electrophoretic-mobility shift assay (EMSA). The results of this combined analysis revealed that violaxanthin significantly inhibited nitric oxide (NO) and the prostaglandin E₂ (PGE₂). Interestingly, violaxanthin effectively inhibited LPS-mediated nuclear factor-κB (NF-κB) p65 subunit translocation into the nucleus, suggesting that the violaxanthin anti-inflammatory activity may be based on inhibition of the NF-κB pathways. In conclusion, violaxanthin of C. ellipsoidea holds promise for use as a potential anti-inflammatory agent for either therapeutic or functional adjuvant purposes.