Background: Antineoplastic agents, including vincristine, often induce neuropathic pain and magnesium deficiency clinically, but the causal link between them has not been determined. No drug is available for treating this form of neuropathic pain. Methods: Injection of vincristine (0.1 mg.kg(-1).day(-1), intraperitoneally, for 10 days) was used to induce nociceptive sensitization, which was accessed with von Frey hairs and the plantar tester in adult male Sprague-Dawley rats. Magnesium-L-threonate was administered through drinking water (604 mg.kg(-1).day(-1)). Extracellular and intracellular free Mg2+ were measured by Calmagite chromometry and flow cytometry. Molecular biologic and electrophysiologic experiments were performed to expose the underlying mechanisms. Results: Vincristine injection induced allodynia and hyperalgesia (n = 12), activated tumor necrosis factor-alpha/nuclear factor-kappa B signaling, and reduced free Mg2+ in cerebrospinal fluid by 21.7 +/- 6.3% (mean +/- SD; n = 13) and in dorsal root ganglion neurons by 27 +/- 6% (n = 11). Reducing Mg2+ activated tumor necrosis factor-a/nuclear factor-kappa B signaling in cultured dorsal root ganglion neurons. Oral application of magnesium-L-threonate prevented magnesium deficiency and attenuated both activation of tumor necrosis factor-alpha/nuclear factor-kappa B signaling and nociceptive sensitization (n = 12). Mechanistically, vincristine induced long-term potentiation at C-fiber synapses, up-regulated N-methyl-D-aspartate receptor type 2B subunit of N-methyl-D-aspartate receptor, and led to peptidergic C-fiber sprouting in spinal dorsal horn (n = 6 each). The vincristine-induced pathologic plasticity was blocked by intrathecal injection of nuclear factor-kappa B inhibitor (n = 6), mimicked by tumor necrosis factor-a, and substantially prevented by oral magnesium-L-threonate (n = 5). Conclusions: Vincristine may activate tumor necrosis factor-a/nuclear factor-kappa B pathway by reduction of intracellular magnesium, leading to spinal pathologic plasticity and nociceptive sensitization. Oral magnesium-L-threonate that prevents the magnesium deficiency is a novel approach to prevent neuropathic pain induced by chemotherapy.