Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that are crucial for adaptation of metazoans to limited oxygen availability. Recently, HIF activation and inhibition have emerged as therapeutic targets in various human diseases. Pharmacologically desirable effects of HIF activation include erythropoiesis stimulation, cellular metabolism optimization during hypoxia and adaptive responses during ischaemia and inflammation. By contrast, HIF inhibition has been explored as a therapy for various cancers, retinal neovascularization and pulmonary hypertension. This Review discusses the biochemical mechanisms that control HIF stabilization and the molecular strategies that can be exploited pharmacologically to activate or inhibit HIFs. In addition, we examine medical conditions that benefit from targeting HIFs, the potential side effects of HIF activation or inhibition and future challenges in this field.