There is growing evidence that uridine may act as an endogenous neuromodulator with a potential signaling role in the central nervous system in addition to its function in pyrimidine metabolism. We previously found that acute morphine treatment significantly increased uridine release in the dorsal striatum of mice, indicating that uridine may contribute to morphine-induced neurobehavioral changes. In the present study, we analyzed the mechanism involved in morphine-induced uridine release and the role of uridine in morphine-induced neurobehavioral changes. Uridine release in the dorsal striatum of mice was assessed by in vivo microdialysis coupled with high performance liquid chromatography (HPLC) after morphine treatment. Western blotting and immunofluorescence were used to evaluate the expression of uridine-related proteins. Morphine-induced neurobehavioral changes were assessed by locomotor activity, behavioral sensitization and conditioned place preference (CPP) test. The expression of NT5E, an extracellular enzyme involved in formation of nucleosides, including uridine, was specifically knocked down in the dorsal striatum of mice using adeno-associated virus (AAV)-mediated short hairpin RNA (shRNA). The results indicated that both acute and chronic morphine administration significantly increased uridine release in the dorsal striatum, and this was associated with upregulation of NT5E but not other uridine-related proteins. Inhibition of NT5E with APCP or shRNA markedly inhibited morphine-induced uridine release in the dorsal striatum and related neurobehavioral changes, including hyperlocomotor activity, behavioral sensitization and CPP. Our data give a better understanding of the contribution of NT5E to morphine-induced uridine release and neurobehavioral changes, and identify NT5E as a potential target for treating morphine abuse.