Objective To observe the expression of endomorphin-2 (EM2) in spinal dorsal horn of bone cancer pain (BCP) rat model and explore its relationship with pain behavior. Methods Adult male SD rats were randomly divided into BCP group, sham operation group and naive group. While Walker-256 cancer cells were injected into the bone marrow cavity of the upper tibia in BCP group. The sham group were treated with the same amount of normal saline. At different time points after cancer cell injection (0d, 3d, 5d, 7d, 10d, 14d, 17d and 21d), the pain behavior of each group was detected, and then EM2 was analyzed by immunohistochemistry and high performance liquid chromatography. In BCP group, the mu-opioid receptor antagonist β-FNA, morphin or EM2 was injected intrathecally when the threshold of paw withdrawal reached the lowest value. Pain behavior was measured immediately after injection. Results Compared with the sham group and naive group, the paw withdrawal threshold of BCP group decreased significantly on the 5th day after injection and reached the lowest value on the 14th day (n=10/group; P＜0.05). In BCP group, the immunohistochemical staining and the expression of EM2 in spinal dorsal horn were significantly decreased (P＜0.05). The decrease of EM2 expression level in BCP group was significantly correlated with the decrease of paw withdrawal threshold (P＜0.001, r=0.963). The analgesic effect of intrathecal injection of EM2 was stronger than that of morphine in BCP group. However, intrathecal injection of β-FNA promoted pain behavior. Conclusion Our findings suggest that the decrease of spinal EM2 contributes to the loss of endogenous analgesia and the enhancement of pain transmission in BCP.