Despite the widespread use of radiotherapy to treat painful bone metastases, the mechanism underlying the analgesic effect of low dose ionizing radiation is unknown. Bone cancer pain is mostly associated with an inflammatory response dominated by local activation of osteoclasts and by astrogliosis in the spinal cord. We determined the effects of a 6 Gy irradiation given focally on osteolytic sarcoma cells inoculated in humeri of mice. Pain behavior was assessed using the rota-rod and the grip force test. Seven days post-irradiation (day 17 post-tumor implantation) the performance of mice markedly improved on the rotarod (non-irradiated, 67±16 s vs irradiated, 223±22 s; P=0.0005), and the grip force test (non-irradiated, 34±4 g vs irradiated, 55±2 g; P=0.001). This improvement was similar to the analgesia achieved with 30 mg/kg of the cyclooxygenase (COX) inhibitor ketorolac (Rota-rod, 67±16 s vs 178±35 s; P=0.01: grip force test, 34±4 g, vs 60±5 g; P=0.003). Following irradiation, the tumor mass and the number of osteoclasts did not decrease while the expression of two pro-inflammatory cytokines (monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-α) increased. Tumor irradiation led to clear differences in the spinal cord. These include a decrease in glial activity (astrocytes and microglial cells) as well as pain mediators such as dynorphin, COX-2 and chemotactic cytokine receptor (CCR2). We conclude that the analgesic effect of low dose irradiation of bone cancer is associated with the alteration of nociceptive transmission in the central nervous system.