The use of iron ions as catalyst of oxidation with hydrogen peroxide, known as the Fenton reaction, is important for industry and biological systems. It has been widely studied since its discovery in the 19th century, but important aspects of the reaction as which is the oxidant, the role of oxygen, and the oxidation state of Fe still remain unclear. In this work new mechanistic insights of the oxidation of carbohydrates by the Fenton reaction using glycerol as experimental model are described. The reaction was studied by means of oxidation reduction potential (ORP) measures. The stoichiometry was measured, showing the important role of oxygen for lowering H2O2 consumption under aerobic conditions. Evidence is provided to demonstrate that in this system Fe2+ generates a catalyst by reacting with a substrate to produce a complex, which gives rise to singlet oxygen after reacting with H2O2. This is the first time that the inhibitor effect of Mg2+ is reported in this reaction, and its participation in the mechanism is described. A rational mechanism for the oxidation of glycerol using the Fenton reaction under these specific conditions is proposed. The role of oxygen, the participation of Fe2+, and the inhibition by Mg2+ are fully demonstrated experimentally.