Background: The induced neural stem cells (iNSCs) held great promises for cell replacement therapy, but iNSCs modulation need improvement. Matrix stiffness could control stem cell fates and might be effective to iNSCs modulations. Here the stiffness of hydrogel matrix on the adhesion, proliferation and differentiation of iNSCs were studied. Methods: Hyaluronic acid (HA) hydrogels with gradient stiffness were prepared. The structure and stiffness of hydrogels were detected by scanning electron microscopy (SEM) and rheological test. iNSCs were generated from human blood mononuclear cells and cultured in the hydrogels. The cell adhesion, proliferation and differentiation on gradient stiffness hydrogels were examined by CCK-8 test and immunofluorescence staining. Results: All hydrogels showed typical soft tissue, with the elastic modulus increasing with concentration (0.6-1.8%), ranging from 17 to 250 Pa. The iNSCs maintained growth and differentiation on all gels, but showed different behaviors to different stiffness. On the softer hydrogels, cells grew slowly at first but continuously and fast for long term, tending to differentiate into neurons; while on the harder hydrogels, cells adhered and grew faster at the early stage, tending to differentiate into glia cells after long term culture. Conclusions: The results suggested that hydrogels stiffness could regulate the key cellular processes of iNSCs. It was important for iNSCs modulation and application in the future.