Novozyme-435-mediated diastereoselective deacylation of one of the two diastereotopic acyloxymethyl groups in 5-O-acyl-4-C-acyloxymethyl-3-O-benzyl-1,2-O-isopropylidene-α-D-ribofuranose has been achieved in quantitative yield. The exclusive selectivity of the lipase for the 5-O-acyl over the 4-C-acyloxymethyl group in the substrate was confirmed by chemical transformation of enzymatically monodeacetylated compound to 1,2-O-isopropylidene-C-4-spiro-oxetanoribofuranose. Further, the selective biocatalytic deacylation methodology has been utilized for the efficient synthesis of C-4'-spiro-oxetanoribonucleosides of uracil (U) and thymine (T) in 37 and 45% overall yields, respectively.