Matrix stiffness regulates epigenetic state and cell reprogramming in a biphasic manner. This study shows that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, with maximum efficiency at an intermediate stiffness of 20 kPa. ATAC sequencing analysis reveals increased chromatin accessibility to neuronal genes at 20 kPa. Histone acetylation and histone acetyltransferase (HAT) activity are highest on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, which shuttle HAT into the nucleus, increase with decreasing matrix stiffness, but reduced importin-9 on soft matrices limits nuclear transport. These factors result in a biphasic regulation of HAT transport into the nucleus, directly demonstrated on matrices with dynamically tunable stiffness. The study reveals a mechanism of mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications. The study investigates how matrix stiffness regulates chromatin reorganization and cell reprogramming. It finds that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, peak levels of histone acetylation and HAT activity are observed on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, which shuttle HAT into the nucleus, increase with decreasing matrix stiffness, but reduced importin-9 on soft matrices limits nuclear transport. These factors result in a biphasic regulation of HAT transport into the nucleus, directly demonstrated on matrices with dynamically tunable stiffness. The findings reveal a mechanism of mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.Matrix stiffness regulates epigenetic state and cell reprogramming in a biphasic manner. This study shows that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, with maximum efficiency at an intermediate stiffness of 20 kPa. ATAC sequencing analysis reveals increased chromatin accessibility to neuronal genes at 20 kPa. Histone acetylation and histone acetyltransferase (HAT) activity are highest on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, which shuttle HAT into the nucleus, increase with decreasing matrix stiffness, but reduced importin-9 on soft matrices limits nuclear transport. These factors result in a biphasic regulation of HAT transport into the nucleus, directly demonstrated on matrices with dynamically tunable stiffness. The study reveals a mechanism of mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications. The study investigates how matrix stiffness regulates chromatin reorganization and cell reprogramming. It finds that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, peak levels of histone acetylation and HAT activity are observed on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, which shuttle HAT into the nucleus, increase with decreasing matrix stiffness, but reduced importin-9 on soft matrices limits nuclear transport. These factors result in a biphasic regulation of HAT transport into the nucleus, directly demonstrated on matrices with dynamically tunable stiffness. The findings reveal a mechanism of mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.