Abstract: Lactobacillus casei 21008 was studied for its ability of producing cholesterol oxidase through cholesterol oxidase indicator plates and by measuring cholesterol oxidase activity. And the ability of its cholesterol-reducting activity in vitro was assessed in MRS-CHO medium and liquid egg yolk. Results indicated that L. casei 21008 was negative for extracellular synthesis of cholesterol oxidase, while observed intracellular cholesterol oxidase-like enzyme, and L. casei 21008 showed 302.7U/L enzyme activity. The reduction of the cholesterol level reached 69.5% in MRS-CHO broth after 18h, which mainly depended on the adsorption and assimilation. Orthogonal experiments in liquid egg yolk estimated that the optimum condition of the factors for cholesterol removal by L. casei 21008 was egg yolk∶water=2∶1 (v/v) , 8% (v/v) inoculum, 30h fermentation. Validation experiments showed that the cholesterol reduction rate was 16.82%, pH5.57.