While the first phase of glycolysis consumes energy to convert glucose to glyceraldehyde 3-phosphate (G3P), the second phase produces energy. The energy is released over a sequence of reactions that turns G3P into pyruvate. The energy-releasing phase—steps 6-10 of glycolysis—occurs twice, once for each of the two 3-carbon sugars produced during steps 1-5.

The first energy-releasing step—considered the 6th step of glycolysis overall—consists of two concurrent events: oxidation and phosphorylation of G3P. The electron carrier NAD⁺ removes one hydrogen from G3P, oxidizing the 3-carbon sugar and converting (reducing) NAD⁺ to form NADH and H⁺. The released energy is used to phosphorylate G3P, turning it into 1,3-bisphosphoglycerate.

In the next step, 1,3-bisphosphoglycerate converts ADP to ATP by donating a phosphate group, thereby becoming 3-phosphoglycerate. The 3-phosphoglycerate is then converted into an isomer, 2-phosphoglycerate.

Subsequently, 2-phosphoglycerate loses a water molecule, becoming the unstable molecule 2-phosphoenolpyruvate, or PEP. PEP easily loses its phosphate group to ADP, converting it into a second ATP molecule and becoming pyruvate in the process.

The energy-releasing phase releases two molecules of ATP and one molecule of NADH per converted sugar. Because it occurs twice—for each 3-carbon sugar produced in the energy-requiring phase of glycolysis—four ATP molecules and two NADH molecules are released. Thus, for each glucose molecule, glycolysis results in a net production of two ATP molecules (4 produced minus 2 used during the energy-requiring phase) and two NADH molecules.

Glycolysis produces two 3-carbon pyruvate molecules from one 6-carbon glucose molecule. In the presence of oxygen, pyruvate can be broken down into carbon dioxide in the Krebs cycle, releasing many ATP molecules. NADH amasses in the cell, where it can be converted back into NAD⁺ and used for further glycolysis.