6-Tuliposides A (6-PosA) and B (6-PosB) are major secondary metabolites in tulip (Tulipa gesneriana), having an acyl group at the C-6 position of d-glucose. They serve as precursors of the antimicrobial -methylene--butyrolactones tulipalins A (PaA) and B (PaB). The conversions of 6-PosA/6-PosB to PaA/PaB are catalyzed by tuliposide-converting enzymes A and B (TCEA and TCEB), respectively. A minor Pos, 1-PosA, which has the acyl group at the C-1 position of d-glucose, has been identified in some wild tulip species, but availability of this compound is limited. Here, by using the TCEs, we established a facile enzymatic process for 1-PosA synthesis from the naturally occurring 1,6-diacyl-glucose type of Pos (PosD and PosF). We first discovered that TCEA and TCEB react preferentially with PosD and PosF, respectively, to form 1-PosA and the corresponding Pa derived from the 6-acyl group, demonstrating that the TCEs specifically acted on the 6-acyl group, but not the 1-acyl group, of the substrates. Using TCEB, 300mg of PosF was completely converted to 1-PosA and PaB in 10min at room temperature. Then, 160mg of 1-PosA (75% molar yield) was purified by column chromatography. This one-step enzymatic process dramatically improves accessibility to 1-PosA.