A vitamin D3 hydroxylase is purified from Pseudonocardia autotrophica cell, and a primer is designed based on amino acid sequence obtained from hydroxylase. Subsequently, PCR is conducted using genomic DNA of Pseudonocardia autotrophica as a template to clone a gene for the vitamin D3 hydroxylase. By conducting a conversion reaction using a microorganism in which the vitamin D3 hydroxylase gene is expressed using a proper expression system, a hydroxide of vitamin D or the like (e.g., hydroxy vitamin D3) can be produced with high efficiency.