Hair follicle stem cells (HFSCs) can quickly activate and migrate to the wound site, differentiating into epidermal stem cells to facilitate early epithelialization. During the wound healing process, microRNAs (miRNAs) function coordinately. Chinese medicine borneol is derived from the Cinnamomum camphora plant, and its principal component, L-borneol, is renowned for its potential in facilitating skin wound healing. However, it remains unclear whether L-borneol can stimulate HFSCs to differentiate into epidermal cells or whether miRNAs are involved in this process. In the current study, HFSCs were isolated from the vibrissae of rats and identified based on the expression of CD34, Integrin-β1 and keratin type 1 cytoskeletal 15(CK15). We observed that stimulation with L-borneol significantly increased the differentiation marker K14 in HFSCs, suggesting that L-borneol could promote the differentiation of HFSCs into basal layer cells. On this basis, we transfected and confirmed that rno-miR-127 inhibitor could promote the differentiation of HFSCs. Furthermore, we demonstrated that PODXL2 is a target gene of rno-miR-127 through dual-luciferase reporter assays and confirmed that the rno-miR-127 mimic could inhibit the expression of PODXL2. To further elucidate the targeting relationship, we constructed the siPODXL2 fragment using siRNA technology, demonstrating that reducing PODXL2 expression can inhibit the differentiation of HFSCs into basal layer cells. Finally, a rat full-thickness skin defect model illustrated L-borneol-mediated negative regulation of PODXL2 by rno-miR-127, promoting skin injury repair through HFSCs.