VD3 is a crucial vitamin for human health, as it enhances calcium absorption in the intestines and prevent rickets. Calcifediol (25(OH)VD3) and calcitriol (1α,25(OH)2VD3) are two derivatives of vitamin D3 that play an important role in preventing and treating osteoporosis, as well as regulating human physiological functions. Currently, the production of calcifediol, and calcitriol primarily relies on chemical synthesis, which has disadvantages such as low product yield, numerous by-products, and environmental unfriendliness. Therefore, developing a green, safe, and environmentally friendly biocatalytic synthesis pathway is of utmost importance. This article mainly reviews the biocatalytic synthesis pathways of calcifediol, and calcitriol. The P450 enzymes, including P450 monooxygenases (cytochrome P450 monooxygenases, CYPs) and P450 peroxygenases (unspecific peroxygenases, UPOs), are crucial for the production of calcifediol and calcitriol. The catalytic mechanism of the extensively studied P450 monooxygenases, the selection of suitable redox partners, and the key residues involved in the enzyme's catalytic activity are analyzed. In addition, the review explores H2O2-driven UPOs, including their catalytic mechanism, strategies for high heterologous expression, and in situ regeneration of H2O2. UPOs are regarded as highly promising biocatalysts because they can facilitate reactions without the need for expensive cofactors and redox partners. This review offers insights into the engineering of P450 for the efficient production of vitamin D3 derivatives.