Abstract: Aim: To determine the proteins that interact with the carboxyl-terminal of the μ opioid receptor (MOR-C) after chronic morphine exposure. Methods: The brain cDNA library of chronic morphine treatment rats was screened using rat MOR-C to investigate the regulator of opioids dependence in the present study. The brain cDNA library from chronic morphine-dependent rats was constructed using the SMART (Switching Mechanism At 5′ end of RNA Transcript) technique. Bacterial two-hybrid system was used to screening the rat MOR-C interacting proteins from the cDNA library. RT-qPCR and immunoblotting were used to determine the variation of MOR-C interacting proteins in rat brain after chronic morphine treatment. Column overlay assays, immunocytochemistry and coimmunoprecipitation were used to demonstrate the interaction of MOR-C and p75NTR-associated cell death executor (NADE)or A20-binding inhibitor of nuclear factor kB (ABIN-1). Results: 21 positive proteins, including 19 known proteins were screened to interact with rat MOR-C. Expression of several of these proteins was altered in specific rat brain regions after chronic morphine treatment. Among these proteins, ABIN-1 and NADE were confirmed to interact with rat MOR-C by in vitro protein–protein binding and coimmunoprecipitation in Chinese hamster ovary (CHO) cells and rat brain with or without chronic morphine treatment. Saturation binding studies showed that ABIN-1 had no effect on MOR binding. However, the interaction of ABIN-1 and MOR inhibited the activation of G proteins induced by DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin). MOR phosphorylation, ubiquitination, and internalization induced by DAMGO were decreased in Chinese hamster ovary cells that coexpressed MOR and ABIN-1. The suppression of forskolin-stimulated adenylylcyclase by DAMGO was also inhibited by the interaction of ABIN-1with MOR. In addition, extracellular signal-regulated kinase activation was also negatively regulated by overexpression of ABIN-1.These data suggest that ABIN-1 is a negative coregulator of MOR activation, phosphorylation, and internalization in vitro. ABIN-1 also inhibited morphine-induced hyperlocomotion in zebrafish larvae (AB strain). By utilization of an antisense morpholino oligonucleotide (MO) gene knockdown technology, the ABIN-1MO-injected zebrafish larvae showed a significant increase (approximately 60%) in distance moved compared with control MO-injected larvae after acute morphine treatment (P≤0.01). Conclusion: Understanding the rat MOR-C interacting proteins and the proteins variation under chronic morphine treatment may be critical for determining the pathophysiological basis of opioid tolerance and addiction. Among these proteins, ABIN-1 negatively regulates MOR function in vitro and in vivo. Other MOR-C interacting proteins’ influence on the opioid tolerance and addiction need further study.