The pyrimidine-specific nucleoside hydrolase Yeik (CU-NH) from Escherichia coli cleaves the N-glycosidic bond of uridine and cytidine with a 102-104-fold faster rate than that of purine nucleoside substrates, such as inosine. Such a remarkable substrate specificity and the plausible hydrolytic mechanisms of uridine have been explored by using QM/MM and MM MD simulations. The present calculations show that the relatively stronger hydrogen-bond interactions between uridine and the active-site residues Gln227 and Tyr231 in CU-NH play an important role in enhancing the substrate binding and thus promoting the N-glycosidic bond cleavage, in comparison with inosine. The estimated energy barrier of 30 kcal/mol for the hydrolysis of inosine is much higher than 22 kcal/mol for uridine. Extensive MM MD simulations on the transportation of substrates to the active site of CU-NH indicate that the uridine binding is exothermic by ∼23 kcal/mol, more remarkable than inosine (∼12 kcal/mol). All of these arise from the noncovalent interactions between the substrate and the active site featured in CU-NH, which account for the substrate specificity. Quite differing from other nucleoside hydrolases, here the enzymatic N-glycosidic bond cleavage of uridine is less influenced by its protonation.