Three-dimensional cell culture is widely used for challenging to biomedical tissue reconstruction. A mass of the reconstructed tissue has been limited because of difficulty in supplying sufficient amount of oxygen deep into the densely cultured cells. Although many methods are available for supplying oxygen, we focused on utilizing microbubbles. The aims of the present study were therefore to develop a microbubble generator suitable for cell culture, and to examine its effects on cellular activity. A new microbubble generator was designed so that it could be autoclaved and settled in a culture vessel with medium. Filtered clean air was introduced to the generator, mechanically sheared by a rotating disk, and then supplied into the culture vessel. This method significantly elevated the dissolved oxygen level by approximately 1.5mg/l. Microscopic measurement showed that 66.8% of the bubbles was distributed within 5-20μm in diameter, meaning that the generated bubbles had a good morphological feature of microbubbles. With and without microbubbles, gel-embedded MC3T3-E1 osteoblastic cells were three-dimensionally incubated for 3 days. Cell viability assay showed that the introduction of microbubbles increased necrotic cell death, which might be due to hyperoxia as well as fluid shearing force. However, relative alkaline phosphatase activity was significantly enhanced by microbubbles. Although further examination is needed, microbubbles would have a potential to enhance osteoblastic cell activity, and could be utilized for effective aeration in dense three-dimensional cell culture.