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Glycosylation, which is catalyzed by UDP-glycosyltransferases (UGTs), is an important biological modification for the structural and functional diversity of ginsenosides. In this study, the promiscuous UGT109A1 from <i>Bacillus subtilis</i> was used to synthesize unnatural ginsenosides from natural ginsenosides. UGT109A1 was heterologously expressed in <i>Escherichia coli</i> and then purified by Ni-NTA affinity chromatography. Ginsenosides Re, Rf, Rh1, and R1 were selected as the substrates to produce the corresponding derivatives by the recombinant UGT109A1. The results showed that UGT109A1 could transfer a glucosyl moiety to C3-OH of ginsenosides Re and R1, and C3-OH and C12-OH of ginsenosides Rf and Rh1, respectively, to produce unnatural ginsenosides 3,20-di-<i>O</i>-β-d-glucopyranosyl-6-<i>O</i>-[α-l-rhamnopyrano-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20<i>S</i>-tetraol (<b>1</b>), 3,20-di-<i>O</i>-β-d-glucopyranosyl-6-<i>O</i>-[β-d-xylopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20<i>S</i>-tetraol (<b>6</b>), 3-<i>O</i>-β-d-glucopyranosyl-6-<i>O</i>-[β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20<i>S</i>-tetraol (<b>3</b>), 3,12-di-<i>O</i>-β-d-glucopyranosyl-6-<i>O</i>-[β<i>-</i>d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20<i>S</i>-tetraol (<b>2</b>), 3,6-di-<i>O</i>-β-d-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (<b>5</b>), and 3,6,12-tri-<i>O</i>-β-d-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (<b>4</b>). Among the above products, <b>1</b>, <b>2</b>, <b>3</b>, and <b>6</b> are new compounds. The maximal activity of UGT109A1 was achieved at the temperature of 40 °C, in the pH range of 8.0–10.0. The activity of UGT109A1 was considerably enhanced by Mg²⁺, Mn²⁺, and Ca²⁺, but was obviously reduced by Cu²⁺, Co²⁺, and Zn²⁺. The study demonstrated that UGT109A1 was effective in producing a series of unnatural ginsenosides through enzymatic reactions, which could pave a way to generate promising leads for new drug discovery.