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Collagen XXIV (Col24α1) is a recently discovered fibrillar collagen. It is known that mouse Col24α1 is predominantly expressed in the forming skeleton of the mouse embryo, as well as in the trabecular bone and periosteum of the newborn mouse. However, the role and mechanism of Col24α1 in osteoblast differentiation and mineralization remains unclear. By analyzing the expression pattern of Col24α1, we confirmed that it is primarily expressed in bone tissues, and this expression gradually increased concomitant with the progression of osteoblast differentiation. Through the use of a lentivirus vector-mediated interference system, silencing Col24α1 expression in MC3T3-E1 murine preosteoblastic cells resulted in significant inhibition of alkaline phosphatase (ALP) activity, cell mineralization, and the expression of osteoblast marker genes such as runt-related transcription factor 2 (Runx2), osteocalcin (OCN), ALP, and type I collagen (Col I). Subsequent overexpression not only rescued the deficiency in osteoblast differentiation from Col24α1 silenced cells, but also enhanced osteoblastic differentiation in control cells. We further revealed that Col24α1 interacts with integrin β3, and silencing Col24α1 up-regulated the expression of Smad7 during osteoblast differentiation while at the same time inhibiting the phosphorylation of the Smad2/3 complex. These results suggest that Col24α1 imparts some of its regulatory control on osteoblast differentiation and mineralization at least partially through interaction with integrin β3 and the transforming growth factor beta (TGF-β) /Smads signaling pathway.