Background: While opioids play a crucial role in pain’s relief, chronic exposure results in tolerance and dependence. Efforts should be made to alleviate the side effect induced by opioids. Many proteins which functionally interact with MOR can regulate the effect of opioids. Our bacterial yeast two-hybrid experiment showed ABIN-1 could bind to MOR. Here, we studied the profile and mechanism of ABIN-1 on morphine tolerance and dependence. Experimental Approach: ABIN-1 in mouse brain was interfered by AAV virus. The tolerance and dependence induced by morphine were assessed in hotplate and conditioned place preference test. The regulation of β-arrestin signalling of MOR was observed in MOR-CHO cell lines after ABIN-1 overexpression. The interaction of proteins was detected by co-immunoprecipitation and immunofluorescence. The expression of proteins was tested by western blotting and immunohistochemistry. Key Results: Morphine tolerance and dependence were attenuated by overexpression of ABIN-1 in mouse brains. ABIN-1 in the hippocampus and nucleus accumbens participated in morphine tolerance and physical dependence. MOR phosphorylation and internalization were weakened by ABIN-1 after opioids treatment. Formation of ABIN-1-β-arrestin-2 complexes promoted the translocation of β-arrestin-2 to the plasma membrane and accelerated its ubiquitination and degradation. Furthermore, attenuation of morphine tolerance by ABIN-1 was abolished in β-arrestin-2 knockout mice. Conclusions and Implications: These findings indicate that ABIN-1 co-operates with β-arrestin2 and MOR to alleviate morphine tolerance and dependence. ABIN-1 may be a target to alleviate morphine tolerance.