Carbonyl sulfide (COS) is one of the major sources of stratospheric sulfate aerosols, which affect the global radiation balance and ozone depletion. COS-degrading microorganisms are ubiquitous in soil and important for the global flux of COS. We examined the sulfur isotopic fractionation during the enzymatic degradation of COS by carbonyl sulfide hydrolase (COSase) from Thiobacillus thioparus THI115. The isotopic fractionation constant (34ɛ value) was -2.2±0.2‰. Under experimental conditions performed at parts per million by volume level of COS, the 34ɛ value for intact cells of T. thioparus THI115 was -3.6±0.7‰, suggesting that, based on Rees' model, the 34ɛ value mainly depended on COS transport into the cytoplasm. The 34ɛ value for intact cells of T. thioparus THI115 was similar to those for Mycobacterium spp. and Williamsia sp., which are known to involve the conserved region of nucleotide sequences encoding the clade D of β-class carbonic anhydrase (β-CA) including COSase. On the other hand, the 34ɛ value was distinct from those for bacteria in the genus Cupriavidus. These results provide an insight into biological COS degradation, which is indispensable for estimating the COS global budget based on the isotope because of the significant contribution of COS degradation by microorganisms harboring β-CA family enzymes.