The resolution of α- and β-anomers of glucopyranosides and galactopyranosides was achieved via enzyme-catalysed regioselective acylation. This two-step procedure to prepare pure α- and β-anomers of glycopyranosides would be most useful for the cases where glycosidases are not available or expensive to purchase. From a synthetic viewpoint, the regioselective acylation of glycopyranosides provides access to mono- and di-esters with well-defined substitution patterns.