In the field of tissue engineering and regenerative medicine, porosity, pore size, and pore interconnectivity of three-dimensional scaffolds affect cellular attachment, proliferation and, migration, and degree of tissue infiltration. In this study, porous chitosan scaffolds with macropores interconnected by micropores were prepared using a thermally induced phase-separation process. Water adsorption properties of scaffolds were investigated by soaking in phosphate-buffered saline (PBS) at room temperature. The chitosan scaffold with macropores interconnected by micropores (CS-10B) adsorbed PBS more rapidly than a chitosan scaffold containing only macropores (CS). An investigation of cell attachment and distribution showed that human dermal fibroblasts (HDFs) culture on chitosan scaffolds in vitro were present on both the surface and cross-sections of the CS-10B scaffold, but only on the surface of the CS scaffold. Studies of tissue responses to the prepared scaffolds, evaluated by subcutaneous implantation in rats, showed that the organization of tissues in the CS-10B scaffold was superior to that in the CS scaffold, and the CS-10B implant degraded faster than the CS implant in vivo. The high interconnectivity of porous scaffolds with macropores interconnected by micropores enhanced biocompatibility and biodegradability, suggesting the excellent potential applications of such chitosan scaffolds in the field of tissue engineering and regenerative medicine.