Chronic morphine-induced antinociceptive tolerance is a major unresolved issue in clinical practices, which is associated with microglia activation in the spinal cord. E3 ubiquitin ligase Pellino1 (Peli1) is known to be an important microglia-specific regulator. However, it is unclear whether Peli1 is involved in morphine tolerance. Here, we found that Peli1 levels in the spinal cord were significantly elevated in morphine tolerance mouse model. Notably, Peli1 was expressed in a great majority of microglia in the spinal dorsal horn, while downregulation of spinal Peli1 attenuated the development of morphine tolerance and associated hyperalgesia. Our biochemical data revealed that morphine tolerance-induced increase in Peli1 was accompanied by spinal microglia activation, activation of mitogen-activated protein kinase (MAPK) signaling, and production of proinflammatory cytokines. Peli1 additionally was found to promote K63-linked ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6) in the spinal cord after repeated morphine treatment. Furthermore, knocking down Peli1 in cultured BV2 microglial cells significantly attenuated inflammatory reactions in response to morphine challenge. Therefore, we conclude that the upregulation of Peli1 in the spinal cord plays a curial role in the development of morphine tolerance via Peli1-dependent mobilization of spinal microglia, activation of MAPK signaling, and production of proinflammatory cytokines. Modulation of Peli1 may be a potential strategy for the prevention of morphine tolerance.