Gly3PDH catalyzes the oxidation/reduction of glycerol-3-phosphate (Gly3P) and dihydroxyacetone phosphate (DHAP).

DHAP + NADH + H⁺  <=> Glycerol-3-Phosphate + NAD⁺

Glycerol is generated by digestion of fats and can be converted to glycerol-3-phosphate by the enzyme glycerol phosphate. Conversion of Gly3P to DHAP by gly3PDH allows all of glycerol's carbon backbone to enter the glycolysis, gluconeogenesis, or pentose phosphate pathways (via conversion of DHAP to glyceraldehyde-3-phosphate).