The mutation of D311 to tyrosine in endo-glycoceramidase II from Rhodococcus sp. and the use of a poorly recognized substrate, 2-chloro-4-nitrophenyl β-cellobioside, have provided appropriate conditions for the efficient synthesis of alkyl β-cellobioside derivatives. The mutant D311Y was characterized by a lowered KM value for the hydrolysis of 2-chloro-4-nitrophenyl β-cellobioside and increased transglycosylation when using aliphatic 1,3-diols or alcohols bearing a δ-hydroxy ketone function as acceptors. Closer analysis revealed that the transglycosylation/hydrolysis ratio in reactions catalyzed by the mutant was completely inversed and weak secondary hydrolysis was postponed, thus providing the basis for high transglycosylation yields (between 68 and 93%). Overall, results confirm that the enhancement of transglycosylation in glycoside hydrolases can be achieved by a combination of destabilized transition states and increased recognition for acceptor molecules.