Cutaneous wounds can be treated using skin substitutes, but they heal with scarring and absence of skin adnexal structures. We previously demonstrated hair follicle neogenesis in dermal-epidermal composites made of neonatal foreskin human keratinocytes and human dermal papilla cells grafted onto nude mice. A challenge to adapting this approach to graft large areas in humans is that dermal papilla cells lose trichogenicity when expanded in vitro. Herein, a peptide derived from a coiled-coil domain of multimerin-1, TSN6, was evaluated for its effects on graft characteristics and hair follicle formation. In a hair follicle reconstitution assay, TSN6 increased the number of hair fibres by 1.8-fold (p value < 0.05). Dermal-epidermal composites, constructed using late-passage human dermal papilla cells and incubated with TSN6 prior to grafting, retained 14 of 14 grafts for 10-12 weeks, whereas scrambled and vehicle groups kept only 9 of 12 and 13 of 16 grafts, respectively. Histological evaluation of skin grafts showed the presence of human hair follicles in 12 of 14 dermal-epidermal composites in the TSN6 group, 3 of 9 in the scrambled group and 6 of 13 in the vehicle group. The median number and interquartile range of hair follicles was 4.5 (1.8, 10.3) for the TSN6 group, 0 (0, 3.5) for the scrambled group and 0 (0, 3.3) for the vehicle group. TSN6 also increased epidermal thickness, showing a thickness of 127 ± 18 μm for the TSN6 group and 70 ± 28 μm and 94 ± 18 μm for the scrambled and vehicle groups, respectively. In summary, TSN6 increases epidermal thickness and promotes hair follicle neogenesis in a skin substitute.