The isomerization of glucose to fructose represents a key intermediate step in the conversion of cellulosic biomass to fuels and renewable platform chemicals, namely, 5-hydroxymethyl furfural (HMF), 2,5-furandicarboxylic acid (FDCA), and levulinic acid (LA). Although both Lewis acids and Brønsted bases catalyze this reaction, the base-catalyzed pathway received significantly less attention due to its lower selectivity to fructose and the poor yields achieved (<10%). However, we recently demonstrated that homogeneous organic Brønsted bases present a similar performance (∼31% yield) as Sn-containing beta zeolite, a reference catalyst for this reaction. Herein, we report on the first extensive kinetic and mechanistic study on the organic Brønsted base-catalyzed isomerization of glucose to fructose. Specifically, we combine kinetic experiments performed over a broad range of conditions (temperature: 80–120 °C; pH 9.5–11.5; reactant: glucose, fructose) with isotopic studies and in situ 1H NMR spectroscopy. Pathways leading to isomerization and degradation of the monosaccharides have been identified through careful experimentation and comparison with previously published data. Kinetic isotope effect experiments were carried out with labeled glucose to validate the rate-limiting step. The ex situ characterization of the reaction products was confirmed using in situ 1H NMR studies. It is shown that unimolecular (thermal) and bimolecular (alkaline) degradation of fructose can be minimized independently by carefully controlling the reaction conditions. Fructose was produced with 32% yield and 64% selectivity within 7 min.