The effect of mustard leaf on nitric oxide (NO) production was investigated using lipopolysaccharide (LPS)-stimulated peritoneal macrophages. LPS induced the production of a large amount of nitrite, an indicator of NO. However, the addition of the ethylacetate (EtOAc) or n-butanol (BuOH) fraction of mustard leaf to LPS-stimulated peritoneal macrophages inhibited excessive production of nitrite significantly. Moreover, compared with no treatment, LPS impaired cell viability significantly. However, the EtOAc fraction of mustard leaf significantly protected cells that had been exposed to LPS. In addition, the result of nitrite production per cell indicated that the mustard leaf fractions significantly suppressed nitrite synthesis by macrophages. In particular, the EtOAc fraction was a stronger inhibitor of nitrite synthesis than the BuOH fraction. As NO is one of the critical mediators in various disorders, these results may help to explain certain pharmacological activities of mustard leaf.