The present invention relates to a polypeptide having 25-hydroxyvitamin D3-1&agr;-hydroxylase activity, being useful for the prevention, diagnosis and therapeutic treatment of adult diseases such as osteoporosis induced by the decrease of active type vitamin D3 and catalyzing the final stage of vitamin D3 activation; and the gene encoding the polypeptide. In accordance with the present invention, the following can be provided; a polypeptide having 25-hydroxyvitamin D3-1&agr;-hydroxylase activity, DNA encoding the polypeptide, a recombinant DNA prepared by inserting the DNA in a vector, a transformant carrying the recombinant DNA, a method for preparing 25-hydroxyvitamin D3-1&agr;-hydroxylase by using the transformant, a method for preparing 1&agr;, 25-dihydroxyvitamin D3 comprising using the polypeptide having 25-hydroxyvitamin D3-1&agr;-hydroxylase activity, and an antibody recognizing the polypeptide.