This study investigates the role of matrix stiffness in regulating chromatin reorganization and cell reprogramming, specifically the conversion of fibroblasts into neurons (iN). The authors found that matrix stiffness acts as a biphasic regulator, with the highest efficiency of iN conversion at an intermediate stiffness of 20 kPa. ATAC sequencing revealed increased chromatin accessibility to neuronal genes at these stiffness levels. Histone acetylation and histone acetyltransferase (HAT) activity were also highest in the nucleus on 20 kPa matrices. Inhibiting HAT activity abolished the effects of matrix stiffness on iN conversion. G-actin and cofilin levels increased with decreasing matrix stiffness, but reduced importin-9 levels on soft matrices limited nuclear transport. These factors result in a biphasic regulation of HAT transport into the nucleus, which was demonstrated using dynamically tunable matrices. The findings suggest a mechano-epigenetic mechanism that could be valuable for disease modeling and regenerative medicine applications.This study investigates the role of matrix stiffness in regulating chromatin reorganization and cell reprogramming, specifically the conversion of fibroblasts into neurons (iN). The authors found that matrix stiffness acts as a biphasic regulator, with the highest efficiency of iN conversion at an intermediate stiffness of 20 kPa. ATAC sequencing revealed increased chromatin accessibility to neuronal genes at these stiffness levels. Histone acetylation and histone acetyltransferase (HAT) activity were also highest in the nucleus on 20 kPa matrices. Inhibiting HAT activity abolished the effects of matrix stiffness on iN conversion. G-actin and cofilin levels increased with decreasing matrix stiffness, but reduced importin-9 levels on soft matrices limited nuclear transport. These factors result in a biphasic regulation of HAT transport into the nucleus, which was demonstrated using dynamically tunable matrices. The findings suggest a mechano-epigenetic mechanism that could be valuable for disease modeling and regenerative medicine applications.