The pentose phosphate pathway (PPP) is a glucose-oxidizing pathway that runs in parallel to upper glycolysis to produce ribose 5-phosphate and nicotinamide adenine dinucleotide phosphate (NADPH). Ribose 5-phosphate is used for nucleotide synthesis, while NADPH is involved in redox homoeostasis as well as in promoting biosynthetic processes, such as the synthesis of tetrahydrofolate, deoxyribonucleotides, proline, fatty acids and cholesterol. Through NADPH, the PPP plays a critical role in suppressing oxidative stress, including in certain cancers, in which PPP inhibition may be therapeutically useful. Conversely, PPP-derived NADPH also supports purposeful cellular generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) for signalling and pathogen killing. Genetic deficiencies in the PPP occur relatively commonly in the committed pathway enzyme glucose-6-phosphate dehydrogenase (G6PD). G6PD deficiency typically manifests as haemolytic anaemia due to red cell oxidative damage but, in severe cases, also results in infections due to lack of leucocyte oxidative burst, highlighting the dual redox roles of the pathway in free radical production and detoxification. This Review discusses the PPP in mammals, covering its roles in biochemistry, physiology and disease.