Giant cell tumor (GCT) of bone is an aggressive non-cancerous tumor, which consists of multi-nucleated osteoclast-like giant cells, stromal cells, and monocytes. It is believed that stromal cells are the neoplastic component of this tumor. Expression of the receptor activator of nuclear factor kappa B ligand (RANKL) in the stromal cells stimulates the monocytes to form giant multi-nucleated osteoclast-like cells, causing bone over-resorption at the tumor site. Previously, our group has reported the up-regulation of RANKL in GCT of bone stromal cells, but the mechanism is unknown. Using stromal cell culture of GCT obtained from patients, we demonstrated the up-regulation of the transcriptional activator CCAAT/enhancer binding protein beta (C/EBPβ). RANKL promoter studies revealed that C/EBPβ over-expression induced RANKL promoter activity in a dose-dependent manner and a CCAAT-box within the region nt −357/−1 contributed to the basal transcription activity, with a possible C/EBPβ binding element in the region nt −460/−358 leading to further induction. Furthermore, we also showed that C/EBPβ bound to the RANKL promoter in GCT stromal cells in vivo by chromatin immunoprecipitation. To conclude, our study has shown that C/EBPβ is a RANKL promoter activator in stromal cells of GCT of bone and we have proposed a model in which C/EBPβ plays an important role in the osteolytic characteristics and pathological causes of GCT of bone. J. Cell. Biochem. 110: 438–446, 2010. © 2010 Wiley-Liss, Inc.