Although CCAAT/enhancer-binding protein β (C/EBPβ) is involved in osteocalcin gene expression in osteoblast in vitro, the physiological importance of and molecular mechanisms governing C/EBPβ in bone formation remain to be elucidated. In particular, it remains unclear whether C/EBPβ acts as a homodimer or a heterodimer with other proteins during osteoblast differentiation. Here, deletion of the C/EBPβ gene from mice resulted in delayed bone formation with concurrent suppression of chondrocyte maturation and osteoblast differentiation. The expression of type X collagen as well as chondrocyte hypertrophy were suppressed in mutant bone, providing new insight into the possible roles of C/EBPβ in chondrocyte maturation. In osteoblasts, luciferase reporter, gel shift, DNAP, and ChIP assays demonstrated that C/EBPβ heterodimerized with activating transcription factor 4 (ATF4), another basic leucine zipper transcription factor crucial for osteoblast maturation. This complex interacted and transactivated osteocalcin-specific element 1 (OSE1) of the osteocalcin promoter. C/EBPβ also enhanced the synergistic effect of ATF4 and Runx2 on osteocalcin promoter transactivation by enhancing their interaction. Thus, our results provide evidence that C/EBPβ is a crucial cofactor in the promotion of osteoblast maturation by Runx2 and ATF4.