Blasticidin A (BcA), an antibiotic produced by Streptomyces, inhibits aflatoxin production without strong growth inhibition toward aflatoxin-producing fungi. During the course of our study on the mode of action of BcA by two-dimensional differential gel electrophoresis (2D-DIGE), we found a decrease in the abundances of ribosomal proteins in Saccharomyces cerevisiae after exposure to BcA. This phenomenon was also observed by treatment with blasticidin S (BcS) or cycloheximide. BcA inhibited protein synthesis in a galactose-induced expression system in S. cerevisiae similar to BcS and cycloheximide. BcS, but not cycloheximide, inhibited aflatoxin production in Aspergillus parasiticus without inhibition of fungal growth, similar to BcA. A decrease in the abundances of aflatoxin biosynthetic enzymes was observed in 2D-DIGE experiments with Aspergillus flavus after exposure to BcA or BcS. These results suggested that protein synthesis inhibitors are useful to control aflatoxin production.