Various Xanthomonas species cause well-known plant diseases. Among various pathogenic factors, the role of α-1,6-cyclized β-1,2-glucohexadecaose (CβG16α) produced by Xanthomonas campestris pv. campestris was previously shown to be vital for infecting model organisms, Arabidopsis thaliana and Nicotiana benthamiana. However, enzymes responsible for biosynthesizing CβG16α are essentially unknown, which limits the generation of agrichemicals that inhibit CβG16α synthesis. In this study, we discovered that OpgD from X. campestris pv. campestris converts linear β-1,2-glucan to CβG16α. Structural and functional analyses revealed OpgD from X. campestris pv. campestris possesses an anomer-inverting transglycosylation mechanism, which is unprecedented among glycoside hydrolase family enzymes.