Blasticidin A, a Streptomyces metabolite, inhibits aflatoxin production in aflatoxigenic fungi. The mode of action of blasticidin A in the inhibition of aflatoxin production was investigated. Blasticidin A inhibited human PTP1B (a protein tyrosine phosphatase) with the IC 50 value of 27 ”M, but not human PP1A (a protein serine/threonine phosphatase). Dephostatin, a known protein tyrosine phosphatase inhibitor, inhibited aflatoxin production without affecting fungal growth. Blasticidin A delayed expression of aflR, which encodes the key regulator of aflatoxin production, and aflatoxin biosynthetic enzyme genes, whereas dephostatin did not affect their mRNA levels. Aspergillus flavus has two genes encoding PTP1B homologs, AfPTP1B-1 and AfPTP1B-2. Blasticidin A inhibited recombinant AfPTP1B-1 more strongly than did dephostatin, and dephostatin inhibited recombinant AfPTP1B-2 much more strongly than did blasticidin A. These results suggest that aflatoxin production is regulated by some protein tyrosine phosphatases, and that protein tyrosine phosphatase inhibitors are potential aflatoxin production inhibitors.