Agrimonia pilosa Ledeb. has long been used for a useful natural agent ameliorating inflammationrelated symptoms in the folk medicine recipe. This study was performed to investigate effects ofAgrimonia pilosa Ledeb.(AP) on the expression of inflammation related genes such as the induciblenitric oxide synthase (iNOS) in macrophage cell line, RAW 264.7 cells. The AP (whole plants) wasextracted with 80% ethanol and sequentially partitioned with solvents in order to increasepolarity. Among the various solvent extracts of AP, the n-butanol (BuOH) fraction showed themost powerful inhibitory ability against nitric oxide (NO) production in lipopolysaccharide(LPS)-induced RAW 264.7 cells without affecting cell viability. Reverse transcriptase-polymerasechain reaction and Western blot analysis revealed that the BuOH fraction provided a primaryinhibitor of the iNOS protein and mRNA expression in LPS-induced RAW 264.7 cells. The DPPHand OH radical scavenging activities of the several fractions of 80% ethanol extracts of APsignificantly increased by EtOAC and BuOH fractions. Thus, the present study suggests that theresponse of a component of the BuOH fraction to NO generation via iNOS expression provide animportant clue to elucidate anti-inflammatory mechanism of AP.