The elicitor-active hexa-P-D-glucopyranosyl-D-glucitol (1), isolated from mycelial walls of Phytophthora megasperma f. sp. glycinea, induces antibiotic phytoalexin accumulation in soybeans. Biological assays of several oligoglucosides revealed that hexa-β-glucoside 2 is the minimum structural element required for high elicitor activity. Synthesis of the oligosaccharides has made considerable progress as a result of the development of glycosidation procedures. However, only a few methodologies directed to the synthesis of oligosaccharide has been developed. Herein we describe an application of a new one-pot glycosidation methodology to the synthesis of an elicitoractive hexaglucoside 2. At first, two types of one-pot glycosidation, linear one-pot glycosidation and branched one-pot glycosidation, are examined. Both one-pot glycosidations provide a new method to construct several glycosidic bonds in one pot reaction, which enables the rapid and efficient synthesis of oligosaccharides. These one-pot glycosidations are based on the ability to control the reactivities of the glycosyl donors 3 and 8 for the linear one-pot glycosidation (Figure-2) and 30 and 38 for the branched one-pot glycosidation (Figure-4) by selecting appropriate activators. Namely, the glycosyl donors X1 in 3 and 30 are selectively activated in the presence of activator A1 to give disaccharides 9 and 37, respectively. Successive activations of X2 in 9 and 38 in the presence of A2, followed by coupling with the glycosyl acceptors 10 and 37 provide the linear trisaccharide 11 and the branched trisaccharide 35, respectively. The results of linear and branched one-pot glycosidations are shown in Table-1 and Figure-5, respectively. Synthesis of the elicitor-active hexaglucoside 2 by the linear one-pot glycosidation was carried out as follows. Initial coupling of glycosyl trichloroimidate 46 with thioglycoside 47 in the presence of a catalytic amount of TMSOTf gave the tetraglucoside. While the phenylthio groups in 47 and the tetraglucoside are stable to TMSOTf activation, addition of a second activators, NIS/TfOH and glycosyl acceptor 48 to the reaction mixture promoted the selective activation of the phenylthio group in the tetraglucoside to give hexaglucoside 49 in 50% yield. Base treatment of hexaglucoside 49, followed by hydrogenation provided the desired hexaglucoside 2 in 94% overall yield. Our studies on the conformation of hexaglucoside 2 in solution based on AMBER force field and GB/SA solvation model revealed that the β-(1-6) linked backbone residue shows a helical structure to which the two branched β-(1-3) glucoses are attached toward outside of the helix. (Figure-8)