The objective of this study is to investigate the influence of shaking culture on the biological functions of cell aggregates incorporating gelatin hydrogel microspheres in terms of the microspheres/cells ratio. The mixture of MC3T3-E1 cells and the microspheres was cultured in the U-bottomed wells of 96-well plate pre-coated with poly (vinyl alcohol) (PVA) to form cell aggregates incorporating microspheres. When incubated in the static or shaking culture, the size of cell aggregates increased with amounts of gelatin hydrogel microspheres but was similar between the two cultures. At the smaller ratio of microspheres to cells, the viability of cell aggregates under the shaking culture was significantly higher than that of static culture. On the other hand, there was no significant difference in the viability between them at the higher ratio. Gelatin hydrogel microspheres enabled to enhance ATP and mitochondrial activities of cell aggregates under the shaking culture. The effect was high at the smaller microspheres/cells ratio. It is concluded that the shaking culture was promising to allow cells to enhance their activities.