Objective: Acceleration of healing process for osseointegration would be desirable. We previously reported that low-intensity pulsed ultrasound (LIPUS) enhanced differentiation of osteoblasts in polystyrene cell culture plates. The purpose of this study was to examine the effects of LIPUS exposure to osteoblasts on a titanium surface. Methods: Bone marrow cells obtained from the femurs of 8-week male SD rats were directly seeded onto titanium disks placed in 12-multi-well cell culture plates with a-MEM supplemented with 15% FBS, 10^-8M dexamethasone, 10 mM Na-b-glycerophosphate and 50 mg/ml L-ascorbic acid 2-phosphate. A LIPUS exposure system for cell culture generated a 2 msec burst of 3.0 MHz sine waves repeated at 100 Hz, was used for this study. The intensity of LIPUS was 40 mW/cm². The cells were exposed to LIPUS underneath the culture plates for 15 min/day from day 3 after primary seeding (LIPUS group). A control group without ultrasound treatment was also cultivated on a titanium surface. Total RNA was extracted form each group and real-time PCR analysis was performed to quantify the osteoblastic gene expressions. The cell proliferation was observed using the WST-8 assay. Morphologic of the mineralized tissue was performed using scanning electron microscopy (SEM). Elemental analyses of calcified tissue were performed using energy dispersive spectroscopy (EDS) and alizarin red S staining. Results: Osteocalcin and osteopontin gene expressions enhanced more in the LIPUS group than in the control group at days 7 and 14 (p<0.01). The cell proliferation in the LIPUS group was accelerated more than in the control group at day 7 (p<0.01). EDS analysis revealed more calcium and phosphate in the LIPUS group. The total area of stained with alizarin red S in the LIPUS group was much than in the control group at days 14 and 21 (p<0.01). Conclusion: LIPUS accelerates differentiation of osteoblasts on titanium surface.