Patients with cancer, especially breast, prostate, and lung cancer, commonly experience bone metastases that are difficult to manage and are associated with bone cancer pain (BCP). Amitriptyline is often used to treat chronic pain, such as neuropathic pain. In the present study, the effects of amitriptyline on the mechanical withdrawal threshold (MWT) and its underlying mechanisms were evaluated in rat models of BCP. Walker 256 rat mammary gland carcinoma cells were injected into the bone marrow cavity of the right tibia of rats to provoke BCP. Then, amitriptyline was intraperitoneally administered twice daily from fifth day after the operation. Rats with bone cancer showed an apparent decline in the MWT at day 11 after Walker 256 cells inoculation. The levels of the glutamate transporter GLAST in the spinal cord dorsal horn decreased remarkably, and the concentration of the excitatory amino acid (EAA) glutamate (Glu) in the cerebrospinal fluid (CSF) increased substantially. Amitriptyline injection could prevent the decline of MWT in BCP rats. In addition, GLAST was upregulated on the glial cell surface, and Glu levels were reduced in the CSF. However, amitriptyline injection could not prevent the BCP-induced reduction in GLAST in the glial cell cytosol, it further downregulated cytosolic GLAST. Amitriptyline had no significant effect on GLAST mRNA expression, and BCP-invoked PKA/PKC upregulation was prevented. Taken together, these results suggest that the intraperitoneal injection of amitriptyline can prevent the decrease of MWT in BCP rats, the underlying mechanisms may be associated with the inhibition of PKA/PKC expression, thus promoting GLAST trafficking onto the glial cell surface and reducing EAA concentrations in the CSF.