It is well recognized that biocatalysis is of special relevance in organic synthesis. Among the enzymes used, lipases have demonstrated a great versatility in enzymatic hydrolysis, transesterification, or aminolysis reactions. Other biocatalysts, such as oxidoreductases and some liases, are also of interest for organic chemists to synthesize compounds that are difficult to obtain by classical chemical procedures. Recently, biotransformations have shown their utility in natural products synthesis, such as in carbohydrates, steroids and nucleosides. Vitamin D, through its hormonally active form 1α,25-dihydroxyvitamin D3, plays an important role in the endocrine system. This metabolite exhibits a much broader spectrum of biological activities than expected. As a result, considerable effort has been made toward the synthesis of structurally related congeners that show interesting pharmacological applications. Most of the analogues prepared have modifications on the upper side chain, more accessible from a synthetic point of view. However, the enzymatic methodology can simplified the chemical routes allowing the synthesis of adequate precursors in a most simple way. We review here, the utility of biotransformations for the preparation of new Vitamin D analogues using different kind of biocatalysts.