Stevioside (ST) and rebaudioside A (Reb A) are 250 times sweeter than sucrose, but their bitterness and low water solubility limit their application. Rebaudioside Aα (Reb Aα) was synthesized using dextransucrase. Reb Aα exhibited 80-fold greater water solubility and lower thermal mass loss than Reb A and had melting point >180 °C. Reb Aα was stable within pH range 1.4-10 and exhibited excellent storage stability in aqueous solution. In vitro digestion revealed that Reb Aα was hydrolyzed into ST and glucose. In vivo, Reb Aα administration did not affect body weight gain, glucose tolerance, energy homeostasis, or liver histology. Also, in the Reb Aα group, inflammatory crown-like structures were not observed in white adipose tissue and uncoupling protein 1 was significantly upregulated in brown adipose tissue. Gut microbiota analysis revealed no significant differences in microbial composition between the control and Reb A or Reb Aα groups.