The purpose of this study was to evaluate the anti-inflammatory and anti-genotoxic activity of branched-chain amino acids (BCAAs) in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophages. BCAAs inhibited LPS-induced NO production, with 100 mM leucine having the most pronounced effect, suppressing NO production by 81.15%. Valine and isoleucine also reduced NO production by 29.65 and 42.95%, respectively. Furthermore, BCAAs suppressed the inducible nitric oxide synthase mRNA expression. Additionally, BCAAs decreased the mRNA expression of interleukin-6 and cyclooxygenase-2 which are proinflammatory mediators. Anti-genotoxic activities of BCAAs were assessed using the alkaline comet assay and valine, isoleucine, and leucine significantly (p < 0.05) decreased tail length of DNA (damaged portion) to 254.8 ± 7.5, 235.6 ± 5.6, and 271.5 ± 19.9 μm compared than positive control H₂O₂ (434.3 ± 51.3 μm). These results suggest that BCAAs can be used in the pharmaceutical or functional food industries as anti-inflammatory agents or anti-cancer agents.