Trehalulose, a rare sucrose isomer, is a dominant sugar in stingless bee honey, with traces of the trisaccharide erlose. Incubating sucrose solutions with macerated stingless bee parts (head, thorax, and abdomen) from , we observed that sucrose isomerization occurs predominantly in the head incubations, with trehalulose constituting 76.2-80.0% of total detected sugar. By contrast, sucrose hydrolysis occurred in stingless bee abdomen incubations, with glucose and fructose observed as 48.6-51.7% and 48.3-49.7%, respectively, of total detected sugar. Incubating glucose/fructose (1:1) solutions with any bee part did not result in trehalulose formation. In addition, by tracing the C isotope-labeled monosaccharide moieties throughout the isomerization from sucrose to trehalulose and erlose, for the first time, the mechanism was established as an enzymatic double displacement reaction. Sucrose acts as a glucose donor giving a β-d-glucosyl enzyme intermediate with fructose release as demonstrated by mixed isotope products. Glucosylation of fructose (inter- or intramolecularly) with isomerization forms trehalulose (favorable), while glucosylation of sucrose forms erlose (less favorable).