Objective To investigate the effect of intrathecal injection of lentiviral vector mediated GlyT2 siRNA on the development of morphine tolerance in bone cancer pain rats. Methods Female adult rats were used to establish bone cancer pain mode (BCP) by injecting Walker 256 breast tumor cell (TCI), normal saline, lentiviral vector mediated GlyT2 siRNA, negative control were intrathecally injected on 7 day post cell infusion. Morphine was intrathecally injected for 7 consecutive days to induce morphine tolerance from day 9 after cell infusion. Before BCP and 7 days after BCP modeling, the MWT was performed to assess the rats’ pain behavioral changes and %MPE was calculated to evaluate the development of morphine tolerance, western blotting was used to detect the expression of GlyT2 protein. Results After TCI, the MWTs of all groups were significantly decreased on day 7, compared with day 1 (P < 0.05). After intrathecal injection of morphine, the %MPE of LV-GlyT2-siRNA was increased significantly compared with other groups (P < 0.05), and the GlyT2 expression was downregulated in the spinal cord of LV-GlyT2- siRNA (P < 0.05). Conclusion Downregulation of GlyT2 can attenuate morphine tolerance of rats with bone cancer pain.