This anion is involved in many metabolic pathways, including the Calvin cycle in plants and glycolysis.

Dihydroxyacetone phosphate lies in the glycolysis metabolic pathway, and is one of the two products of breakdown of fructose 1,6-bisphosphate, along with glyceraldehyde 3-phosphate.

DHAP is also the product of the dehydrogenation of L-glycerol-3-phosphate, which is part of the entry of glycerol (sourced from triglycerides) into the glycolytic pathway.

Conversely, reduction of glycolysis-derived DHAP to L-glycerol-3-phosphate provides adipose cells with the activated glycerol backbone they require to synthesize new triglycerides.

DHAP also has a role in the ether-lipid biosynthesis process in the protozoan parasite Leishmania mexicana.