Royal sun medicinal mushroom Agaricus brasiliensis KA21 has various beneficial pharmacological effects; however, these effects are influenced by its cultivation conditions. A. brasiliensis KA21 is rich in β-glucan, which promotes wound healing. This study evaluated the wound healing-promoting effects of A. brasiliensis KA21 and the influence of cultivation conditions on these effects. Upon evaluation of its effect on wound healing, the outdoor-cultivated A. brasiliensis KA21 (KAOD) promoted wound healing in mice, and the effect of KAOD was stronger than that of the indoor-cultivated one (KAID). In addition, A. brasiliensis KA21 promoted the synthesis of collagen I and III, which are involved in promoting wound healing; KAOD strongly induced collagen III production. Furthermore, KAOD suppressed the decrease in skin elasticity after acetone application in our mouse model, which was not observed for KAID. These results show that KAOD is useful as a supplement in surgery and injury healing for humans and animals, improving immunity against pathogens in wound areas and promoting wound healing. In addition, KAOD may be useful in the field of skin aesthetics for repairing skin damage and maintaining skin elasticity.