Tricholoma matsutake is popular in Asian countries because of its edibility and medicinal use. T. matsutake is a precious natural medicinal fungus, and it is widely used in food and biological products. This study aimed to explore the mechanism of T. matsutake on promoting proliferation of human immortalized keratinocyte (HaCaT) cells and accelerating wound healing in mice. The MTT assay was used to test the effects of three different T. matsutake extracts (0, 62.5, 125, 250, 500, and 1000 μg/mL) on HaCaT cell viability. HaCaT cells were treated with the three T. matsutake extracts (100, 500 μg/mL) and morphological and biophysical properties were detected by atomic force microscopy with JPK data processing. Western blot analysis detected Notch signaling pathways of HaCaT cells treated with 50% ethanol extract of T. matsutake (50%T) for 24 h (100, 500 and 1000 μg/mL). Mouse wounds were treated with 50%T for 15 days. Wound healing effects were observed on the back skin of mice at different times. The quality of wound healing was estimated by histological staining (hematoxylin and eosin and Masson's trichrome). All data were counted by GraphPad Prism 5 software. The increased concentration of T. matsutake remarkably promoted HaCaT cell proliferation. The Young's modulus of HaCaT cells showed the biggest increase from 1.73 ± 0.13 kPa (0 μg/mL) to 4.57 ± 0.16 kPa (500 μg/mL) in the 50%T group. The Notch1/Jagged1 pathways were upregulated with an increase in concentration (0, 100, 500, and 1000 μg/mL). Moreover, compared with the negative and positive control groups, T. matsutake promoted wound healing in mice by epidermal regeneration, subepidermal tissue formation, and collagen deposition. The results showed that T. matsutake promotes not only proliferation of HaCaT cells but also wound healing in mice.