Mechanical cues arising from extracellular matrices greatly affect cellular properties, and hence, are of significance in designing biomaterials. In this study, a DNA crosslinked hydrogel was employed to examine cellular responses of spinal cord neurons to substrate compliances. Using DNA as crosslinkers in polymeric hydrogel formation has given rise to a new class of hydrogels with a number of attractive properties (e.g., reversible gelation and controlled crosslinking). Here, it was demonstrated that by varying length of crosslinker, monomer concentration, and level of crosslinking, DNA gel stiffnesses span from approximately 100 Pa to 30 kPa. Assessment of neurite outgrowth on functionalized DNA gels showed that although primary dendrite length is not significantly affected, spinal cord neurons extend more primary dendrites and shorter axons on stiffer gels. Additionally, a greater proportion of neurons have more primary dendrites and shorter axons on stiffer gels. There is a pronounced reduction in focal adhesion kinase (FAK) when neurons are exposed to stiffer substrates, suggesting its involvement in neuronal mechanosensing and neuritogenesis in response to stiffness. These results demonstrate the importance of mechanical aspects of the cell-ECM interactions, and provide guidance for the design of mechanical properties of bio-scaffolds for neural tissue engineering applications.