Aim Aconitine and its structurallyrelated diterpenoid alkaloids have been shown to interact differentially with neuronal voltagedependent sodium channels and be responsible for their analgesia and toxicity.Bulleyaconitine A (BAA or BLA) is an aconitine analog and has been prescribed for the management of pain.The present study aimed to evaluate the inhibitory effects of BAA on pain hypersensitivity and morphine antinociceptive tolerance, and explore whether the release of dynorphin A from spinal microglia was associated with its mechanism of actions.Methods Rat models of neuropathic pain, formalin test and bone cancer pain were used, and spinal dynorphin A level and expression were measured.Sample size of animals was six in each study group.Resultes A single intrathecal or subcutaneous (but not intraventricular or local) injection of BAA blocked spinal nerve ligationinduced painful neuropathy, bone cancerinduced pain and formalininduced hyperalgesia by 60% ~ 100%with the ED50 values of 94 ~ 126 ng/rat (intrathecal) and 42 ~ 59 μg · kg1 (subcutaneous), respectively.Following chronic treatment, BAA did not induce either selftolerance to antinociception or crosstolerance to morphine antinociception, and completely prevented morphine tolerance.Spinal BAA antinociception, but not neurotoxicity, was completely blocked by the specific microglial inhibitor minocycline.In a minocyclinesensitive and lidocaineor ropivacaineinsensitive manner, BAA stimulated the release of dynorphin A from the spinal cord, and the primary culture of microglia but not from neurons or astrocytes.The blockade effects of BAA on nociception and morphine tolerance were completely blocked by the specific dynorphin A antiserum and/or κopioid receptor antagonist.Conclusions Our results demonstrated that BAA eliminated pain hypersensitivity and morphine tolerance through the direct stimulation of dynorphin A release from spinal microglia, which was not dependent on the interactions with sodium channels.