In folklore medicine, Acorus calamus has been used as a wound-healing agent for thousands of years; however, there have been few scientific reports on this activity so far. Now, we explored deeply the wound-healing effect of aqueous extracts from the fresh roots and rhizomes of A. calamus in vivo, as well as anti-inflammatory activity in vitro, so as to provide scientific evidence for the traditional application. The wound-healing effect was determined by the image analysis techniques and the histological analysis in the excisional wounding test, and the anti-inflammatory activity was evaluated by the real-time RT-PCR techniques in the lipopolysaccharide-induced RAW 264.7 cells test. Aqueous extracts, administered topically at the dose range from twice to thrice in a day, could enhance significantly the rate of skin wound-healing. Moreover, the extracts could effectively inhibit the mRNA expressions of inflammatory mediators induced by lipopolysaccharide in RAW 264.7 cells. These results showed significantly the wound-healing activity of aqueous extracts in the animal model of excise wound healing, and anti-inflammatory activity in vitro.