A computational model for human transketolase was proposed, showing that thiamine diphosphate activation was based on His110 in place of His481 reported in yeast transketolase. In addition, a complete catalytic reaction pathway was investigated using d-xylulose-5-phosphate and d-ribose-5-phosphate as substrates, showing at every step a perfect superimposition of our model with high-resolution crystallographic structures 3MOS, 4KXV, and 4KXX. This study shows that H2N4′ of the active thiamine diphosphate “V form” no longer has a self-activating role but allows self-stabilization of the cofactor and of the Breslow intermediate. These advances in our knowledge of the human transketolase mechanism offer interesting prospects for the design of new drugs, this enzyme being involved in several diseases, and for a better understanding of the reactions catalyzed by transketolases from other sources.