Protease inhibitors of small molecular size are useful as medical drugs and biological reagents. In the course of our screening of fungi as protease inhibitors, we found Aspergillus oryzae ATCC 20386 to be a mold producing a specific inhibitor, named kojistatin A, against various cysteine proteases. In this paper, we report the isolation, structure, and inhibitory activities of kojistatin A against some proteases. A new cysteine protease inhibitor, named kojistatin A, was found in the extract of a solid culture of Aspergillus oryzae ATCC 20386. The structure of kojistatin A was determined on the basis of chemical and spectroscopic evidence. Kojistatin A specifically inhibited cysteine proteases such as papain, ficin, and bromelain.