Bone homeostasis is orchestrated by the balance of bone formation and resorption. Recent advances have revealed that bone homeostasis could be locally and systemically modulated by immune-related genes and inflammatory cytokines, as osteoclasts and immune cells are originally derived from bone marrow. We previously showed that estrogen receptor-binding fragment-associated antigen 9 (EBAG9) is an immune-related gene that negatively modulates host tumor immunity. Namely, xenografted tumor growth in Ebag9KO mice was impaired due to the increased infiltration of CD8+ T cells, with enhanced activity of degranulation and cytotoxicity (Oncogenesis, 3, e126, 2014). The present study aims to characterize the role of EBAG9 in the bone metabolism. Skeletal structure of Ebag9KO and wild-type (WT) female mice were labeled with fluorochromes tetracycline and calcein. Dual-energy X-ray absorptiometry (DEXA) analysis revealed that femoral bone mineral density (BMD) in Ebag9KO mice was decreased compared with that in WT mice. Microcomputed tomography (μCT) analysis was performed at the distal femur using a μCT35 instrument and three-dimensional representations were reconstructed based on the two-dimensional images from the contoured regions. Ebag9KO mice exhibited decreases in trabecular bone volume (BV/TV) and trabecular thickness (Tb.Th). Similarly, bone morphometric analysis of tibia revealed that BV/TV and Tb.Th in Ebag9KO mice were decreased compared with those in WT mice. Evaluated mechanical strength of femoral bones by a three-point bending test revealed that the peak load were decreased in Ebag9KO mice compared with that in WT mice. Overall, impaired activity of Ebag9 in mice exhibit reduced bone volume and BMD. Based on our findings, we assume that EBAG9 would be a novel modulatory factor that plays a role in the interaction between the bone and immune systems.