Prolyl hydroxylases (PHD) are key regulators of the HIF pathway. These enzymes, acting as oxygen sensors, mediate oxygen-dependent hydroxylation of key proline residues of the HIF-1 α subunit: Pro⁴⁰² and Pro⁵⁶⁴. These modifications allow the von Hippel-Lindau (VHL) tumour suppressor protein to bind to HIF-1 α, leading to its ubiquitination by the E3 ubiquitin ligase complex, and subsequently its degradation within the proteasome. Hydroxylation by prolyl hydroxylases requires the presence of molecular oxygen (O₂). Hypoxic conditions therefore prevents HIF-1 α from being hydroxylated and finally degraded, which allows it to fulfill its role of transcriptional activator.

The PHD family is composed of three isoforms encoded by three independent genes. It has been shown that PHD2 is the main regulator of HIF-1 α low levels in normoxia ⁷, while PHD3 is mainly expressed in hypoxia to inhibit HIF-activated genes when reoxygenation occurs ⁸. PHD1 is thought to have a higher role on HIF-2 α than on HIF-1 α ⁸. A fourth isoform of prolyl hydroxylase has been identified ⁹ which unlike the three others is located within the endoplasmic reticulum. Although its structure and localisation make it resemble collagen prolyl hydroxylases, its action has been shown to be equivalent to the other three HIF-related ones.