Low-molecular-weight proteinase inhibitors of microbial origin have received increasing attention due to their utility in investigating the physiological and/or pathogenic roles of proteinases in vivo and potential therapeutic applications. In this study, we screened for thiol proteinase-specific inhibitors from microorganisms and found that strain EY-21 (identified as Bacillus cereus based on morphological and physiological properties) produces a novel inhibitor named thiolstatin, which specifically inhibits various thiol proteinases. The strain was aerobically cultured at 30°C in a medium containing 1% meat extract, 1% polypepton, 1% glucose, and 0.3% NaCl (pH 7.2). Thiolstatin was purified from 90 liters of culture filtrate via Amberlite XAD-2 column chromatography (eluted with 80% aqueous methanol), Amberlite IRC-50 (NH₄ form) column chromatography (eluted with 0.1 N HCl), and preparative reversed-phase high performance liquid chromatography (LiChroprep RP-18 column with methanol gradient elution in phosphate buffer), yielding four active components: thiolstatin A, B, C, and D. Physicochemical characterization showed that thiolstatins are soluble in water and methanol but insoluble in acetone, ethyl acetate, and ether; they are stable in acidic or neutral solutions but less stable in alkaline conditions, and exhibit specific thin-layer chromatography behavior (Rf values of 0.40 and 0.63 in two solvent systems) and chemical reactivity (positive Sakaguchi and Rydon-Smith reactions, negative ninhydrin, thiosulfate, and 2,3,5-triphenyltetrazolium chloride reactions). The structure of thiolstatin D was determined via identification of acid/alkaline hydrolysis products and analysis of ¹H-NMR and ¹³C-NMR spectra. Inhibitory spectrum studies revealed that thiolstatins potently inhibit thiol proteinases (e.g., papain, ficin, bromelain) with low ID₅₀ values, but have no or weak inhibitory effects on other enzyme types including serine proteinases (except weak inhibition of trypsin), metalloproteinases, carboxyl proteinases, peptidases, and non-proteolytic thiol enzymes (e.g., lactate dehydrogenase, glucose-6-phosphate dehydrogenase), confirming their specificity for thiol proteinases. Additionally, thiolstatins strongly inhibit in vitro fertilization of hamster eggs, a effect not observed with the thiol proteinase inhibitor E-64. These findings indicate that thiolstatins are valuable reagents for elucidating the physiological and pathological roles of thiol proteinases in vivo.