Nicotine has been reported to prolong the wound healing; however, we showed that the topical application of 10−4 M nicotine promoted murine wound healing. The objective of this study was to explore the wound healing effects of nicotine in combination with collagen scaffold using skin defects in rabbit. Three full-thickness skin defects 8 mm in diameter were made on the rabbit auricle. Artificial dermis was applied to the defects, and 10 μl of nicotine solution (10−5, 10−4, and10−3 M), bFGF solution (0.5 μg/10 μl), and both bFGF and 10−4 M nicotine solutions were injected into the artificial dermis once daily for 7 days. Rabbits were sacrificed on day 10, 15, or 20, and the wound healing process was evaluated. bFGF was superior in the formation of the dermis-like tissue and capillaries. In nicotine groups, the epithelial length and the dermis-like tissue formations in the 10−4 M group were superior, in contrast, those were inhibited in the 10−3 M group. The synergistic effect of bFGF and 10−4 M nicotine was not confirmed. This study suggests that the topical application of 10−4 M nicotine promoted wound healing in rabbit, but the effect was not apparent compared with murine models.