A phloem-sap based diet requires unique enzymatic skills for regulating the osmotic pressure differences in the gut lumen between the sucrose-rich ingested sap and the body fluids, which can lead to desiccation and death. In the phloem-feeding whitefly Bemisia tabaci, members of the glycoside hydrolase family 13 (GH13) are likely to play a critical role in mitigating this risk by oligomerizing and isomerizing the products of sucrose hydrolysis. So far, however, the identities of the family members acting as sucrose isomerases (mainly isomerizing trehalulose) remain unknown. In this study, we focused on four putative sucrose isomerase coding genes of B. tabaci that belong to the GH13 family. The four coding genes were selected based on their phylogeny, expression patterns, and motif analyses. We used artificial diets of sucrose and dsRNA to knockdown the expression of each of the target genes and analyzed the sugar composition of the secreted honeydew. We found that in all cases, gene silencing resulted in a significant reduction of the fractions of trehalulose accompanied by an increase in the fractions of the trisaccharide melezitose, suggesting that the insects attempt to compensate for the inability to isomerize sucrose by synthesizing oligosaccharides. In addition, we found that the downregulation of the target genes also resulted in a significant increase in the mortality rates of the silenced insects and a significant delay in the development of their progeny. Taken together, these findings demonstrate the importance of the osmoregulation-by-isomerization strategy in B. tabaci.