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In this study, the conversion of vitamin D 3 (VD 3 ) to its two active forms 25(OH)VD 3 and 1α, 25(OH) 2 VD 3 was carried out by engineering the hydroxylase CYP105A1 and its redox partners Fdx and Fdr. CYP105A1 and Fdx-Fdr were respectively expressed in E. coli BL21(DE3) and purified. The electron transport chain Fdx-Fdr had higher selectivity for the coenzyme NADH than NADPH. HPLC analysis showed that CYP105A1 could hydroxylate the C25 and C1α sites of VD 3 and convert VD 3 to its active forms. Finally, a one-bacterium-multi-enzyme system was constructed and used in whole-cell catalytic experiments. The results indicated that 2.491 mg/L of 25(OH)VD 3 and 0.698 mg/L of 1α, 25(OH) 2 VD 3 were successfully produced under the condition of 1.0% co-solvent DMSO, 1 mM coenzyme NADH and 35 g/L biocatalyst loading. This study contributes to a basis for the industrial production of active VD 3 in future., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)