Na Lian. ATF4 is an osteoblast-enriched transcription factor that regulates Osteocalcin transcription in vitro and osteoblast terminal differentiation in vivo. However, little is known about regulators that interact with ATF4 and affect its activity. In this study, we found that Vimentin, a member of type III intermediate filament proteins, interacted with ATF4 and inhibited its binding to its cognate element, OSE1, on the Osteocalcin promoter. Consequently, activation of Osteocalcin gene transcription by ATF4 was blocked when Vimentin was co-transfected in reporter assays. Overexpressing Vimentin stably in an osteoblast cell line, MC3T3, led to a delay in differentiation. Conversely, knocking down the expression of Vimentin in these cells stimulated osteoblast differentiation. Furthermore, the expression of Vimentin mRNA and protein was high in early osteoblasts but decreased in mature osteoblasts, suggesting that Vimentin down-regulation may be a prequisit to the initiation of osteoblast differentiation. Our studies identify a novel mechanism for an intermediate filament protein to inhibit cell differentiation through a direct interaction with a transcription factor.