Provided is a process for industrially producing cis-3-hydroxy-L-proline useful as a raw material for medicines or as an additive to foods. L-proline is converted into cis-3-hydroxy-L-proline in the presence of an enzyme source which is derived from a microorganism belonging to the genus Streptomyces or Bacillus and which catalyzes hydroxylation of L-proline into cis-3-hydroxy-L-proline, a divalent iron ion and 2-ketoglutaric acid, in an aqueous medium, and the produced cis-3-hydroxy-L-proline is recovered from the aqueous medium. Also provided is a novel enzyme L-proline-3-hydroxylase, which is useful for the process.