Blasticidin S, a protein synthesis inhibitor, inhibits aflatoxin production of Aspergillus flavus without affecting fungal growth. Analysis of metabolites in blasticidin S-treated A. flavus using quadrupole time-of-flight liquid chromatography-mass spectrometry showed that blasticidin S was metabolized into a novel metabolite, N-acetyldeaminohydroxyblasticidin S. Conversion of blasticidin S to N-acetyldeaminohydroxyblasticidin S via deaminohydroxyblasticidin S or N-acetylblasticidin S was observed in in vivo and in vitro A. flavus systems. Blasticidin S and N-acetylblasticidin S inhibited the growth of Aspergillus niger strongly and weakly, respectively, but deaminohydroxyblasticidin S and N-acetyldeaminohydroxyblasticidin S did not inhibit its growth. On the other hand, deaminohydroxyblasticidin S sustained the inhibition of aflatoxin production whereas N-acetylblasticidin S and N-acetyldeaminohydroxyblasticidin S did not. These results suggest that the free amino group at C-13 of blasticidin S and deaminohydroxyblasticidin S may be important for the inhibitory activity of aflatoxin production.