To investigate the inhibitory effect and mechanism of EphB1-Fc targeted blocking EphB receptor-mediated signal on chronic inflammatory pain. Methods A rat model of inflammatory pain was established by intraplantar injection with 50 μl CFA in the left hind paw. The rats were randomly divided into Control group, CFA group and CFA+EphB1-Fc group. Spontaneous pain (flinches) and mechanical pain threshold (PWT) were used to detect the changes of pain behavior of rats. Immunofluorescence and Western blotting were used to detect the changes in the level of EphB signal and inflammatory factors in spinal cord. Results Intrathecally injection of EphB1-Fc significantly alleviated CFA induced hyperalgesia in rats. EphB1-Fc injection obviously inhibited the activation spinal inflammatory factor IL-1β. EphB1-Fc blocked EphB1/SRC/NF-κB signal pathway. Conclusion Blocking EphB1 mediates SRC/NF-κB signal decreases spinal inflammation and alleviates pathological pain