Background Opioid-primed relapse is a global burden. Although current strategies have improved, optimal therapy is urgently needed. Methods A recombinant adenovirus (Ad-NEP) expressing beta-endorphin (beta-EP) was designed and injected intracerebroventricularly (icv) into the right lateral ventricle in rats. Spatial and temporal beta-EP expression in the lateral ventricle wall, subventricular zone and adjacent choroid plexus and the beta-EP concentration in the cerebrospinal fluid (CSF) were observed during a 21-day period. A morphine priming-induced conditioned place preference (CPP) rat model was established. The beta-EP-ir neuron counts, CSF beta-EP concentration, and CPP score, which were used to evaluate morphine-primed reinstatement following extinction, were recorded 7 days after the icv injection. Additionally, the rats were pretreated with the irreversible mu opioid receptor antagonist beta-funaltrexamine (beta-FNA) and the selective kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI) to identify the receptor-dependent mechanism. Results Both peak beta-EP expression in target neurons and the peak CSF beta-EP concentration occurred 7 to 8 days after Ad-NEP icv injection. The sustainable increase in the CSF beta-EP concentration was correlated with a decrease in the CPP score 7 days after the Ad-NEP icv injection. Furthermore, reinstatement was almost reversed by beta-FNA pretreatment 24 hours before the behavioral test, but nor-BNI had little effect. Conclusion The increasing cerebrospinal fluid beta-endorphin concentrations showed that the therapeutic effect on opioid relapse occurred predominantly through a mu opioid receptor-dependent mechanism. The Ad-NEP adenovirus can be considered an alternative therapy for opioid relapse.