Receptor activator of nuclear factor kappa B ligand (RANKL) expression in osteoblasts is regulated by 1,25-dihydroxyvitamin D3 (1,25D3). CCAAT/enhancer-binding protein beta (C/EBPβ) has been proposed to function as a transcription factor and upregulate RANKL expression, but it is still uncertain how C/EBPβ is involved in 1,25D3-induced RANKL expression of osteoblasts. 1,25D3 stimulation increased the expression of RANKL and C/EPBβ genes in osteoblasts and enhanced phosphorylation and stability of these proteins. Moreover, induction of RANKL expression by 1,25D3 in osteoblasts was downregulated upon knockdown of C/EBPβ. In contrast, C/EBPβ overexpression directly upregulated RANKL promoter activity and exhibited a synergistic effect on 1,25D3-induced RANKL expression. In particular, 1,25D3 treatment of osteoblasts increased C/EBPβ protein binding to the RANKL promoter. In conclusion, C/EBPβ is required for induction of RANKL by 1,25D3. [BMB Reports 2019; 52(6): 391-396].