In this study, a new type of polydimethylsiloxane-modified chitosan (PMSC) amphiphilic hydrogel was developed as a soft substrate to explore cellular responses for dermal reconstruction. The hydrogel wettability, mechanical stiffness and topography were controllable through manipulation of the degree of esterification (DE) between hydrophobic polydimethylsiloxane (PDMS) and hydrophilic N,O-(carboxymethyl)-chitosan (NOCC). Based on microphase separation, the incorporation of PDMS into NOCC increased the stiffness of the hybrid through the formation of self-assembled aggregates, which also provided anchor sites for cell adhesion. As the DE exceeded 0.39, the size of the PDMS-rich aggregates changed from nanoscale to microscale. Subsequently, the hierarchical architecture resulted in an increase in the tensile modulus of the hybrid gel up to fourfold, which simultaneously provided mechano-topographic guidance and allowed the cells to completely spread to form spindle shapes instead of forming a spherical morphology, as on NOCC (DE = 0). The results revealed that the incorporation of hydrophobic PDMS not only impeded acidic damage resulting from NOCC but also acted as an adhesion modification agent to facilitate long-term cell adhesion and proliferation on the soft substrate. As proved by the promotion on long-term type-I collagen production, the PMSC hybrid with self-assembled mechano-topography offers great promise as an advanced scaffold material for use in healing applications.