A new class of inhibitors for β-D-glycoside hydrolases, in which a single α-(14)-glycosidic bond is incorporated into an otherwise all-β-(14)-linked oligosaccharide, is described. Such mixed β/α-linkage cellooligosaccharides are not transition-state mimics, but instead are capable of utilising binding energy from numerous subsites, spanning either side of the catalytic centre, without the need for substrate distortion. This binding is significant; a mixed α/β-D-tetrasaccharide acts competitively on a number of cellulases, displaying inhibition constants in the range of 40–300 μM. Using the Bacillus agaradhaerens enzyme Cel5A as a model system, one such mixed β/α-cellooligosaccharide, methyl 4II,4III-dithio-α-cellobiosyl-(14)-β-cellobioside, displays a Ki value of 100 μM, an inhibition at least 150 times better than is observed with an equivalent all-β-linked compound. The three-dimensional structure of B. agaradhaerens Cel5A in complex with methyl 4II,4III-dithio-α-cellobiosyl-(14)-β-cellobioside has been determined at 1.8 Å resolution. This confirms the expected mode of binding in which the ligand, with all four pyranosides in the 4C1chair conformation, occupies the −3, −2 and +1 subsites whilst evading the catalytic (−1) subsite. Such “by-pass” compounds offer great scope for the development of a new class of β-D-glycoside hydrolase inhibitors.