Objective To investigate the effect and mechanism of dexmedetomidine (Dex) on hyperalgesia in morphine withdrawal mice. Methods According to drug treatments, healthy male C57/BL mice were randomly divided into blank control group, morphine withdrawal hyperalgesia model group (M group), M+Dex group, M+minocycline (Min) group, M+Dex+Min group, M+MK8825 group, and M+Min+MK8825 group (n=6). No intervention was given to mice in the blank control group. The other 6 groups of mice were intraperitoneally injected with morphine twice a day at 8:00 a.m. and 6:00 p.m. for 6 consecutive days, followed by naloxone injection to establish the morphine withdrawal hyperalgesia model. The M group received no more drugs, while the M+Dex, M+Min, M+Dex+Min, M+MK8825, and M+Min+ MK8825 groups were given Dex diluted with artificial cerebrospinal fluid, microglia activation inhibitor Min, Dex and Min mixture, calcitonin gene-related peptide (CGRP) inhibitor MK8825, and Min and MK8825 mixture through intrathecal catheter 30 min before naloxone administration, respectively. Mechanical pain thresholds were tested by von Frey. The expression of microglia activation marker ionized calcium-binding adapter molecule 1 (IBA-1) in the spinal dorsal was observed by immunofluorescence and Western blotting. The expression of CGRP protein and mRNA in the spinal cord of each tissue was detected by Western blotting and polymerase chain reaction (PCR). The levels of inflammatory factors tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in the spinal cord were determined by microdialysis. Finally, the effect of Dex on spontaneous inhibitory postsynaptic current (sIPSC) in the spinal dorsal horn was observed by electrophysiology. Results The morphine withdrawal hyperalgesia model was successfully established. Compared with the blank control group, the mechanical pain threshold in the M group was significantly decreased (P<0.05), and the expression of IBA-1, CGRP and the levels of TNF-α and IL-1β in the spinal dorsal horn were significantly increased (all P<0.05). Compared with the M group, the pain thresholds in the M+Dex, M+Min and M+Dex+Min groups were significantly increased (all P<0.05), and the expression of IBA-1, CGRP and the levels of TNF-α and IL-1β in the spinal dorsal horn were significantly decreased (all P<0.05). The levels of TNF-α and IL-1β in spinal dorsal horn of the M+MK8825 group were significantly lower than those of the M group (both P<0.05), while there were no significant differences between the M+MK8825 group and M+Min+MK8825 group (both P>0.05). Electrophysiology results showed that Dex enhanced the amplitude and frequency of sIPSC in the spinal dorsal horn neurons compared with artificial cerebrospinal fluid perfusion (both P<0.05). Conclusion Dex relieves hyperalgesia in morphine withdrawal syndrome by inhibiting spinal microglia activation, reducing the expression of CGRP, alleviating spinal inflammatory response, and enhancing spinal inhibitory electrical activity.