Artificial substrates supporting the healing of skin wounds require specific structural and chemical architectures that promote a recapitulation of the complexity of the native organ. Bottom-up fabrication technologies are emerging as effective strategies to fine tune biochemical, morphological, and structural features intended for regenerative applications. Here, we proposed an electrophoretic replica deposition (EPrD) approach to realize chitosan three-dimensional structures specifically designed to treat patients with serious cutaneous damages or losses. The EPrD process has been optimized to consistently obtain random porosity vs. hierarchical lattice structures, showing mechanical properties in the range of skin tissue (E = 0.2–20 MPa). The obtained patches were tested in vivo via a one-stage grafting procedure in a full thickness skin wound rat model. Chitosan patches showed no adverse reactions throughout the experimental period (14 days). Hair follicles and sebaceous glands were observed in histological sections, indicating the regeneration of a thin epidermal layer with more skin appendages. Immunohistochemistry results demonstrated that keratin 10 was mostly expressed in basal and suprabasal layers, like normal skin, in structures with random porosity and with smaller lattice structures. The obtained results show the potential of EPrD to innovate the design of artificial substrates in skin healing therapies.