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Aside from suitable flap transplantation, there is no effective method to prevent the development of soft tissue defects after the occurrence of full-thickness skin defects. Even bilayered cultured skin is too thin to ameliorate soft tissue defects. Therefore, the construction of an adipocyte-containing hypodermis under a skin substitute is required. The aim of this study was to investigate the adipogenic properties of a small intestinal submucosa (SIS) scaffold containing adipose-derived stem cells (ASCs) in vitro and in vivo as a potential tool for soft tissue engineering, serving as an adipocyte-containing hypodermis. A series of acellular processes were performed to prepare an SIS scaffold that lacked cellular components and cellular debris. The SIS scaffold induced less severe inflammation at 1, 2, and 4 weeks after subcutaneous implantation than the positive control. ASCs proliferated more rapidly when cultured on the SIS scaffold than when cultured without a scaffold. Furthermore, ASCs were strongly adherent to the scaffold and evenly distributed in the interior of the scaffold. Assessment of the adipogenic properties of the SIS scaffold showed that ASCs could differentiate into adipocytes in vitro and in vivo and that the number of adipocytes in the cell-matrix scaffold group was much greater than that in the pure scaffold group in vivo. Our results suggest that ASCs can differentiate into adipocytes displaying adipogenic potential in the SIS scaffold in vitro and in vivo. These findings demonstrate the potential utility of ASCs combined with the SIS scaffold for the generation of an adipocyte-containing hypodermis that can serve as a more complete skin substitute.