25-hydroxyvitamin D-3 has attracted considerable attention due to its great medical value and huge market demand in animal husbandry. Microbial production of 25-hydroxyvitamin D-3 has been recognized as an alternative superior to traditional chemical synthesis. In this study, a Gram-positive bacteria zju 4-2 (CCTCC M 2019385) was isolated from the soil using vitamin D-3 as the sole carbon source and was identified as Bacillus cereus according to its physiological characteristics and 16S rRNA analysis, which also showed a relatively high capacity for 25-hydroxyvitamin D-3 production. Through systematic optimization of different catalytic conditions, the optimal solvent system of vitamin D-3, vitamin D-3 addition time and concentration, temperature, and pH were shown to be propylene glycol/ethanol (v/v = 9:1), early stationary phase, 2 g/L, 37 degrees C, and pH 7.2, respectively. With these optimal conditions, 796 mg/L of 25-hydroxyvitamin D-3 was achieved after 48 h bioconversion with zju 4-2 at the shake flask level. Finally, up to 830 mg/L 25-hydroxyvitamin D-3 with a yield of 41.5% was obtained in a 5 L fermentation tank. Our developed biotransformation process with this newly isolated strain provides a platform to produce 25-hydroxyvitamin D-3 efficiently at industrialization scale.