Anthrax lethal factor (LeTx) is a critical virulence factor in toxin-challenged cells, as lethal factor (LF) cleaves mitogen-activated protein kinase kinases (MKKs), inhibiting their activity. The physiological importance of this cleavage for macrophage cytolysis remains unclear, because similar proteolysis has been also observed in LeTx-resistant macrophages. Here, we analyzed in vitro proteomic profiles of Raw264.7 lysates treated with LF. In our experiments, neuronal NO synthase (nNOS) was found to be a fragment, suggesting that LF may act on nNOS cleavage. A similar cleavage of nNOS was shown in LeTx-challenged HEK293 cells expressing nNOS by a transient transfection. However, the cleavage site on nNOS is a unique leader sequence among the NOS family and this LF-mediated cleavage was not observed in iNOS, a major NOS isoform for anti-bactericidal NO production, even though NO level in LeTx-challenged cells was dramatically reduced. Our findings suggest that LF is directly capable of cleaving cellular protein(s) other than MKKs, and that these actions potentiate to promote the cytotoxic mechanisms of anthrax.