Objective The aim of this study is to investigate the effect of matrix stiffness changes on the differentiation of neural stem cells (NSCs) and the underlying mechanism.Methods A rat model of spinal cord injury was constructed. Polyacrylamide gel substrates with different stiffness (0.7 kPa, 40 kPa) were prepared, and were cultured with primary rat NSCs. NSCs cells were transfected with piezo type mechanosensitive ion channel component 1 (Piezo1) shRNA plasmid. Immunofluorescence staining was used to detect the percentage of positive cells for the neuron marker doublecortion(DCX) and the astrocyte marker GFAP. Immunohistochemistry experiment and Western blot were used to detect the protein expression of Piezo1.Results The results showed that compared with the 0.7 kPa group, the number of DCX-positive cells in the 40 kPa group increased, while the number of GFAP-positive cells decreased, as well as the protein expression of Piezo1 increased. The expression of Piezo1 was significantly up-regulated in the injured tissue of rats with spinal cord injury compared with the sham group. Silencing of Piezo1 reverse the effect of 40 kPa matrix stiffness on NSCs proliferation, as indicated by decreased number of DCX-positive cells, and increased number of GFAP-positive cells increased. Further studies found that knockdown of Piezo1 leads to decreased expression of collagen IV and fibronectin. Recombinant fibronectin reversed the effect of sh-Piezo1 on the differentiation of NSCs.Conclusion Rigid base stiffness regulates the differentiation of NSCs by promoting the expression of Piezo1 protein and up-regulating the expression of type IV collagen and fibronectin. This study provides a new perspective for the treatment of spinal cord injury based on biomaterials.