Activating transcription factor 4 (ATF4) is an osteoblast-enriched transcription factor that regulates osteocalcin transcription and osteoblast terminal differentiation. To identify functional partners of ATF4, we applied ROS17/2.8 osteoblast nuclear extracts and purified recombinant His-ATF4 onto a Ni(+) affinity matrix chromatography column. Vimentin was identified by liquid chromatography-mass spectrometry. Coimmunoprecipitation and pulldown assays revealed that vimentin interacted with ATF4 with its first leucine zipper domain. DNA cotransfection and gel retardation demonstrated that vimentin inhibited the transactivation activity of ATF4 on osteocalcin by preventing it to bind OSE1, the ATF4 binding site on the osteocalcin promoter. Northern hybridization revealed that vimentin was expressed at a high level in immature osteoblasts and a low level in fully differentiated osteoblasts. Down-regulation of vimentin by small interfering RNA induced endogenous osteocalcin transcription in immature osteoblasts. Conversely, ectopic overexpression of vimentin in osteoblasts inhibited osteoblast differentiation as shown by lower alkaline phosphatase activity, delayed mineralization, and decreased expression of osteoblast marker genes such as bone sialoprotein and osteocalcin. Together, our data uncover a novel mechanism whereby a cytoskeletal protein, vimentin, acts as a break on differentiation in immature osteoblasts by interacting with ATF4.