4-Hydroxyproline (4Hyp) is crucially important for the collagen triple helix formation and its thermal stability. It is catalyzed by collagen prolyl 4-hydroxylases (C-P4Hs) that are α₂β₂ tetramers of two identical catalytic α-subunits (encoded by P4HA1, P4HA2 and P4HA3) and two β-subunits that are protein disulfide isomerases (PDI). The functions of isoenzymes I and II (P4HA1 and P4HA2) are widely studied but very little is currently known about P4HA3. Here we show that P4ha1 is generally the most abundant isoform in mouse, whereas P4ha3 expression is tissue and developmental stage specific. P4ha3 expression is highest in the developing skeletal system and peaks at E12.5 together with the other isoforms. Postnatal P4ha3 expression is low and mainly present in calvaria, lung, heart and muscle. The P4ha3 expression virtually disappears at or after 1 week of age in all tissues studied. To evaluate the P4HA3 function in collagen synthesis, we produced cells that lack P4HA3 (P4ha3^-/-) and cells that have P4HA3 as the only isoform (P4ha1^-/-;P4ha2^-/-) by CRISPR/Cas9. C-P4H activity assays indicated no 4Hyp formation in type I collagen by the P4ha1^-/-;P4ha2^-/- cells, suggesting that the remaining isoform P4HA3 does not participate in type I collagen synthesis. The P4ha3^-/- cells secreted intact pepsin-resistant collagen that had similar 4Hyp content and melting temperature as WT collagen. Secreted collagen also assembled into supramolecular collagen fibrils. In conclusion, our results provide new insights into collagen biosynthesis and suggest that prolyl 4-hydroxylation of type I collagen is mediated by P4HA1 and P4HA2 but not by P4HA3.