Substrate stiffness affects cell migration and spreading. Our study revealed that the stiffness of the cell-adhesive substrate affected the migration pattern of neural cells. We observed the migration of neural cells differentiated from neurosphere-forming neural stem/progenitor cells (NSPCs) on collagen gels with various degrees of stiffness achieved by chemical cross-linking. Both glial and neuronal cells broadly spread and migrated when stiff collagen gels (G'=5.5kPa, G″=0.2kPa) were used as the substrate. In contrast, the migration of glial cells was suppressed within the limited area on the soft collagen gels (G'=0.8kPa, G″=0.2kPa). Filopodia were rarely observed in glial cells on the soft collagen gels. Analysis of the intercellular distance between the closest neural cells after differentiation from NSPCs indicated that glial cells more broadly spread on the stiff collagen gels than on the soft gels. Immunocytochemical analysis showed that most of the migrated cells were glial cells, suggesting that migration of glial cells was dependent on the stiffness of substrate.