Density functional theory was used to study the thermodynamics and kinetics for the glycosidic bond cleavage in deoxyuridine. Two reaction pathways were characterized for the unimolecular decomposition in vacuo. However, these processes are associated with large reaction barriers and highly endothermic reaction energies, which is in agreement with experiments that suggest a (water) nucleophile is required for the nonenzymatic glycosidic bond cleavage. Two (S(N)1 and S(N)2) reaction pathways were characterized for direct hydrolysis of the glycosidic bond by a single water molecule; however, both pathways also involve very large barriers. Activation of the water nucleophile via partial proton abstraction steadily decreases the barrier and leads to a more exothermic reaction energy as the proton affinity of the molecule interacting with water increases. Indeed, our data suggests that the barrier heights and reaction energies range from that for hydrolysis by water to that for hydrolysis by the hydroxyl anion, which represents the extreme of (full) water activation (deprotonation). Hydrogen bonds between small molecules (hydrogen fluoride, water, or ammonia) and the nucleobase were found to further decrease the barrier and overall reaction energy but not to the extent that the same hydrogen-bonding interactions increase the acidity of the nucleobase. Our results suggest that the nature of the nucleophile plays a more important role in reducing the barrier to glycosidic bond cleavage than the nature of the small molecule bound, and models with more than one hydrogen fluoride molecule interacting with the nucleobase provide further support for this conclusion. Our results lead to a greater fundamental understanding of the effects of the nucleophile, activation of the nucleophile, and interactions with the nucleobase for this important biological reaction.