Collagen is one of the most abundant proteins in humans and plays an essential role in cell maintenance and organization. Collagen has a unique triple-helix structure composed of three polypeptides, and hydroxylation of proline residues is vital for the stability of this triple-helix. Prolyl-4-hydroxylase (P4H) is an enzyme with an α2β2 tetramer arrangement that functions to post-translationally hydroxylate proline residues in the collagen chain. The α subunit (P4Hα) of the P4H α2β2 tetramer contains the catalytic site, whereas the β subunit (PDI) maintains the solubility and activity of the α subunit. In this study, two plasmids were used to produce an active human P4H tetramer (rhP4H) in Pichia pastoris. The P4Hα was cloned into pPICZαA, and PDI was cloned into PHIL. Real-time PCR showed that the P4H gene was expressed at the mRNA level. SDS-PAGE and western blotting analysis showed that the secreted expression of rhP4H was achieved successfully in P. pastoris GS115 cells. The catalytic activity of rhP4H was tested in a system that contained O2, Fe2+, α-ketoglutaric acid and ascorbic acid. Liquid chromatography coupled with tandem mass spectrometry was used to monitor the hydroxylation of proline. The results showed that rhP4H specifically hydroxylated proline residues only in the Gly-X-Y structure but did not react with free proline. We hypothesize that rhP4H can be engineered to control structural properties of recombinant collagen by selecting a certain degree of hydroxylation of proline residues, which should contribute to creating engineered collagens with specific end uses.