The effects of substrate material on the spatio-temporal behavior of cells is an important issue. Although cell aggregation has been observed on various fibroin substrates, the mechanisms of this aggregation have yet to be fully clarified. In this study, cell aggregation behavior on fibroin substrates were evaluated, focusing on the distance between each cell and the direction of individual cell migration. Our results showed that on fibroin substrates cells did not attract each other. However cells stayed close to adjacent cells over 24 hours of cultivation.