By culturing mouse macrophages in vitro, The inhibitory effect of L-NNA on the synthesis nitric oxide (NO) was investigated, which was induced by IFN-γ in macrophages. It was found that L-NNA could inhibit the synthesis of NO, the inhibition was related to L-NNA dose, and L-arginine could reverse the inhibition. The results suggest that L-NNA likely competitively combined with the activate site of inducible nitric oxide synthase (iNOS), that inhibited the synthesis of NO from L-arginine pathway in macrophage.