Morphine is a well-known mu-opioid receptor (MOR) agonist and an efficient analgesic, but its long-term use inevitably leads to drug addiction and tolerance. Here, we show that specific inhibition of mu-arrestin2 with its siRNA lentivirus microinjected in mice periaqueductal gray matter (PAG) significantly improved both acute and chronic morphine analgesia and delayed the tolerance in the hotplate test. The specific effect of beta-arrestin2 was proven by overexpression or knockdown of its homology beta-arrestin1 in PAG, which showed no significant effects on morphine analgesia. These findings suggest that specific siRNA targeting beta-arrestin2 may constitute a new approach to morphine therapy and other MOR agonist-mediated analgesia and tolerance.