Background: Nitric oxide (NO) plays a key role in regulating various biological processes including vasodilation, neurotransmission, inflammatory responses, the immune system and apoptosis. In endothelial cells, the production of NO is known to be enhanced in response to mechanical stimulation through intracellular calcium-mediated activation of endothelial nitric oxide synthase (eNOS). In this study, we investigated whether low-intensity ultrasound is capable of mechanically stimulating endothelial cells, which leads to an increase in intracellular calcium and enhancement of NO production. Methods and Results: Cultured bovine aortic endothelial cells (BAECs) were loaded with Calcium Green-1 AM, a calcium-sensitive fluorescent dye, and then exposed to low-intensity ultrasound for 1 min. During the exposure, an increase in fluorescence intensity was observed by laser scanning confocal microscopy. This result indicated that lowintensity ultrasound acted as a mechanical stimulus and induced a transient increase in intracellular calcium in BAECs. To measure the change in NO production, cultured BAECs were exposed to low-intensity ultrasound for 0 (control), 30 and 120 min while loaded with NO-sensitive fluorescent indicator dye DAF-2DA. The fluorescence intensities of the BAECs obtained by laser scanning confocal microscopy were increased in proportion to the exposure time, suggesting that lowintensity ultrasound enhanced NO production in the cells. Conclusions: Low-intensity ultrasound can mechanically stimulate BAECs, resulting in an enhancement of NO production through the activation of the intracellular calcium signaling pathway. Further studies are needed to elucidate the mechanism by which ultrasound increases NO production at a molecular level.