The objective of this study was to investigate the effects of latent TGF-β binding protein 4 (LTBP-4) on elastic fiber regeneration in three-dimensional cultures of human dermal fibroblasts (HDFs). Appropriate collagen scaffold for elastic fiber regeneration was also examined. Collagen sponges cross-linked at 120 °C and composed of small pores (25 μm on average) was favorable for elastic fiber regeneration by HDFs. Addition of LTBP-4, followed by culture for 21 days, accelerated elastic fiber accumulation within the scaffolds. Conditioned scaffolds containing either HDFs or LTBP-4-built mature elastic fibers were implanted between the dermis and the cutaneous muscle of mice. The combined use of HDFs and LTBP-4 resulted in thicker tissues containing elastic fibers. These results indicate that weakly cross-linked collagen sponges can be used as scaffolds for regenerating elastic fibers both in vitro and in vivo, and that the addition of LTBP-4 accelerates the deposition of both elastin and fibrillin-1, and increases cell proliferation. These techniques may be useful for generating cutaneous or cardiovascular tissue equivalents; furthermore, they may serve as a useful method for the three-dimensional analyses of drugs used to treat skin diseases or to examine the microstructure of elastin networks.