The attachment of sugar to flavonoids enhances their solubility. Glycosylation is performed primarily by uridine diphosphate-dependent glycosyltransferases (UGTs). The UGT from Bacillus cereus, BcGT-1, transferred three glucose molecules into kaempferol. The structural analysis of BcGT-1 showed that its substrate binding site is wider than that of plant flavonoid monoglucosyltransferases. In order to create monoglucosyltransferase from BcGT-1, the error-prone polymerase chain reaction (PCR) was performed. We analyzed 150 clones. Among them, two mutants generated only kaempferol O-monoglucoside, albeit with reduced reactivity. Unexpectedly, the two mutants harbored mutations in the amino acids located outside of the active sites. Based on the modeled structure of BcGT-1, it was proposed that the local change in the secondary structure of BcGT-1 caused the alteration of triglucosyltransferase into monoglucosyltransferase.