Background: Cancer pain is a complex medical syndrome. Understanding its underlying mechanisms relies on the use of animal models which can mimic the human condition. A crucial component of this model is the quantity of tumor cells; however, the exact relationship between the doses of tumor cells on bone cancer pain is yet unknown. Objective: We explored the relationship of different doses of Walker 256 carcinoma cells using a bone cancer pain model in rats, and evaluated its success and stability. Study Design: Experimental animal study using a comparative design. Setting: Experimental Animal Center and Tumor Institute of Traditional Chinese Medicine. Methods: We constructed the bone cancer pain model by implanting Walker 256 carcinoma cells into the right tibia of Sprague-Dawley (SD) rats (150 – 170 g). Spontaneous pain, mechanical threshold, and paw withdrawal latency (PWL) were measured and x-ray, bone mineral density (BMD), histological, interleukin-1 beta (IL-1β) mRNA, carboxyterminal telopeptide of type I collagen (ICTP), and bone alkaline phosphatase (BAP) were analyzed for bone pain model evaluation. Results: The results showed that: (1) the 3 doses (3×105 , 3.5×105 , 4×105 ) of Walker 256 carcinoma cells can induce bone cancer pain from day 7 to day 21 after implantation into the right tibia of SD rats; (2) compared to the control group, 3×105 , 3.5×105 , and 4×105 Walker 256 carcinoma cells produced different pain manifestations, where the 3.5×105 dose of Walker 256 carcinoma cells resulted in the greatest bone cancer pain response; (3) the 3.5×105 dose induced the lowest mortality rate in rats; (4) Walker 256 carcinoma cells (3×105 , 3.5×105 , and 4×105 ) resulted in a significant decrease in the general condition and body weight of rats, where the 3.5×105 and 4×105 doses of carcinoma cells produced a greater effect than 3×105 dose of carcinoma cells; (5) progressive spontaneous pain, PWL, and mechanical threshold were exacerbated by 3.5×105 and 4×105 doses of carcinoma cells; (6) implantation of 3.5×105 and 4×105 doses of carcinoma cells induced progressive bone destruction and decrease in BMD; (7) ICTP and BAP were significantly increased following the implantation of 3.5×105 and 4×105 doses of carcinoma cells; (8) IL-1βmRNA was significantly up-regulated in the spinal cord of rats implanted with 3.5×105 and 4×105 doses of carcinoma cells. Limitations: One limitation of this study was the small sample size; therefore, additional research is needed to provide better validation. Another limitation is the unavailability of small animal Micro computed tomography (CT), which is a more advanced and precise technique in determining bone marrow density than the x-ray imaging system we used. In addition, ethology experiments during late-stage tumor progression can be more objective. Conclusion: This study provides evidence that implantation of 3.5×105 and 4×105 dose of Walker 256 carcinoma cells produced the greatest effects in relation to the bone cancer pain model in SD rats, and 3.5×105 dose induced the lowest mortality rate. Key words: Bone cancer pain model, Walker 256 carcinoma cells, different doses