Gly3PDH catalyzes the oxidation of glycerol-3-phosphate (Gly3P) to dihydroxyacetone phosphate (DHAP). Electrons are transferred to NAD⁺, forming NADH in the reaction. Glycerol is generated by digestion of fats. Conversion of Gly3P to DHAP by Gly3PDH allows all of glycerol's carbon backbone to enter the glycolysis, gluconeogenesis, or pentose phosphate pathways (via glyceraldehyde-3-phosphate).

Gly3P + NAD⁺ <=> DHAP + NADH + H⁺ 

Gly3PDH participates in the dihydroxyacetone phosphate/glycerol-3-phosphate shuttle of insect muscle (Figure 15.11)