Bacillus subtilis SFF34 degrading cholesterol was applied to reduce residual cholesterol content in fermented flatfish. When the bacterial cells were inoculated as a start culture, a maximal level (1.7 U/g) of cholesterol oxidase was obtained after 10 days, which was two times higher than that (0.8 U/g) without inoculation. Residual cholesterol contents with and without inoculation of the cells were 0.5 mg/g and 0.8 mg/g after 12 days of fermentation, respectively. Cholesterol derivatives including cholesterol- 5${alpha},;6{alpha}$-epoxide, 4-cholesten-3-one and 7${eta}$-hydroxycholesterol were detected in raw flatfish as well as fermented flatfish. Campesterol and 25-hydroxycholesterol were detected only after fermentation. However, no significant differences in their contents were observed regardless of inoculation.