Isolated and purified L-proline-4-hydroxylase which catalyzes hydroxylation of L-proline at the 4-position of L-proline in the presence of 2-ketoglutaric acid and a divalent iron ion to produce trans-4-hydroxy-L-proline. The L-proline-4-hydroxylase may be isolated from a microorganism selected from the group consisting of Dactylosporanlium sp. RHI deposited with the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology, Japan, under the designation PERM BP-4400 and Amycolatopsis sp. RH2 deposited as FERM BP-4581.