Prolyl 4-hydroxylases (P4Hs) have central roles in the synthesis of collagens and the regulation of oxygen homeostasis. The 4-hydroxyproline residues generated by the endoplasmic reticulum (ER) luminal collagen P4Hs (C-P4Hs) are essential for the stability of the collagen triple helix. Vertebrate C-P4Hs are alpha2beta2 tetramers with three isoenzymes differing in their catalytic alpha subunits. Another P4H family, the HIF-P4Hs, hydroxylates specific prolines in the alpha subunit of the hypoxia-inducible transcription factor (HIF), a master regulator of hypoxia-inducible genes, and controls its stability in an oxygen-dependent manner. The HIF-P4Hs are cytoplasmic and nuclear enzymes, likewise with three isoenzymes in vertebrates. A third vertebrate P4H type is an ER transmembrane protein that can act on HIF-alpha but not on collagens. All P4Hs require Fe2+, 2-oxoglutarate, O2, and ascorbate. C-P4Hs are regarded as attractive targets for pharmacological inhibition to control excessive collagen accumulation in fibrotic diseases and severe scarring, while HIF-P4H inhibitors are believed to have beneficial effects in the treatment of diseases such as myocardial infarction, stroke, peripheral vascular disease, diabetes, and severe anemias. Studies with P4H inhibitors in various animal models of fibrosis, anemia, and ischemia and ongoing clinical trials with HIF-P4H inhibitors support this hypothesis by demonstrating efficacy in many applications.