Periodontitis is a destructive disease that is likely the result of the activities of different microbial complexes, including anaerobic Gram-negative periodontopathic bacteria. Butyric acid (sodium butyrate; BA) is a major metabolic by-product of anaerobic Gram-negative periodontopathic bacteria present in subgingival plaque. This study was undertaken to examine the effect of BA on the expression of osteogenesis-related transcription factors and mineralized nodule formation in osteoblastic ROS17/2.8 cells. The cells were cultured with 0 (control), 10(-5), 10(-4), or 10(-3) M BA for up to 7 days. The gene and protein expression levels of transcription factors such as Runx2, Osterix, Dlx5, Msx2, and AJ18, as well as extracellular matrix proteins such as bone sialoprotein (BSP) and osteocalcin, were examined using real-time PCR and Western blotting, respectively. Mineralized nodule formation was detected by alizarin red staining. The expression of Runx2, Osterix, Dlx5, and Msx2 decreased significantly in the presence of 10(-3 )M BA compared to the control, whereas AJ18 expression increased significantly. Mineralized nodule formation decreased markedly in the presence of 10(-3) M BA. Alkaline phosphatase activity and the expression of bone sialoprotein and osteocalcin decreased significantly in the presence of 10(-3) M BA compared to the control. These results suggest that 10(-3) M BA suppresses osteoblastic differentiation and mineralized nodule formation in ROS17/2.8 cells.