There were few studies investigating the effects of the mechanical stimulation provided by daily low-intensity pulsed ultrasound (LIPUS) treatment. LIPUS is known to accelerate bone mineralization and regeneration; however, the precise cellular mechanism is unclear. Our purpose was to determine how daily LIPUS treat-ment affected cell viability, alkaline phosphatase activity, osteogenesis-related gene expression, and mineralized nodule formation in osteoblasts. The typical osteoblastic cell line ROS 17/2.8 cells were cul-tured in the absence or presence of LIPUS stimulation. Daily LIPUS treatments (1.5 MHz; 20 min) were admi-nistered at an intensity of 30 mW/cm2 for 14 days. Expression of osteogenesis-related genes was examined at mRNA levels using real-time polymerase chain reac-tion and at protein levels using weste blotting analy-sis. LIPUS stimulation did not affect the rate of cell viability. Alkaline phosphatase activity was increased after 10 days of culture with daily LIPUS stimulation. LIPUS significantly increased the expression of mRNAs encoding Runx2, Msx2, DIx5, osterix, bone sialoprotein,and bone morphogenetic protein-2, whereas it signifi-cantly reduced the expression of mRNA encoding the transcription factor AJ18. Mineralized nodule for-mation was markedly increased on Day 14 of LIPUS stimulation. LIPUS stimulation directly affected osteo-genic cells, leading to mineralized nodule formation. LIPUS is likely to have a fundamental influence on key functional activities of osteoblasts in alveolar bone.