<p>Fentanyl and its analogues are the most commonly used synthetic opioid analgesics in clinical practice, but their abuse is a significant concern. Drug-paired environmental cues often trigger memory retrieval, leading to relapse, complicating treatment and overdose prevention. In this study we investigated μ-opioid receptor-related molecular mechanisms underlying the retrieval of fentanyl contextual addiction memory in mice. A conditional place preference (CPP) model was established in mice by citrate injections of fentanyl (0.1 mg/kg) for 4 days. By performing whole-brain screening using c-Fos immunofluorescence staining, we found that the paraventricular thalamus (PVT) was dramatically activated. We conducted Western blotting, co-immunoprecipitation and proteomics to evaluate the proteins interacting with μ-opioid receptors on the membrane, and found marked externalization of μ-opioid receptors on the membrane in PVT neurons. We revealed that μ-opioid receptors trafficking in PVT was regulated by the extent of binding of Ap2a1 to the membrane μ-opioid receptors. By conditional knockdown and chemogenetic manipulation, we demonstrated the contribution of μ-opioid receptors to the retrieval of fentanyl contextual memory via modulating the neuronal activity in PVT. In conclusion, this study suggests that Ap2a1-mediated trafficking of μ-opioid receptors underlies the retrieval of fentanyl contextual addiction memory through regulating the neuronal activity in PVT.</p>