Opioids are widely used in clinical practice because of their strong analgesia. However, their use is restricted by such factors as tolerance and opioid-induced hyperalgesia (OIH), so it is critical to find ways to reduce the dosage of opioids to avoid the side effects. In this study, we demonstrated for the first time the regulatory role of A20 in morphine analgesia. By overexpressing and knocking down A20 in the spinal cord of mice, we found that A20 enhanced morphine analgesia rather than tolerance. Then, at the cellular level, different methods were used to confirm that A20 could not only strengthen the inhibition of cAMP induced by opioids drugs, but also affect mu opioid receptor (MOR) and ERK phosphorylation. In addition, we found that A20 interacted with MOR inhibitory protein beta-arrestin2, which could be enhanced by MOR agonists. Furthermore, there was evidence that A20 could inhibit beta-arrestin2 recruitment. Collectively, our results indicated that A20 in the spinal cord could enhance morphine analgesia and increase MOR function through beta-arrestin2. Upregulating A20 expression may be a potential strategy to improve the therapeutic profile of opioids and reduce their side effects. (C) 2020 Elsevier Inc. All rights reserved.