This study aimed to explore the therapeutic potential of umbilical mesenchymal stem cell-derived apoptotic vesicles (UMSC-apoVs) in a 5-Fluorouracil (5-FU)-induced impairment in skin wound healing. UMSC-apoVs were isolated from UMSCs using differential centrifugation after the induction of apoptosis. A murine model was established by administering 5-FU via intravenous tail injection, followed by full-thickness skin wound creation. Mice received local injections of UMSC-apoVs at the lesion site. Wound healing was evaluated based on wound closure rates, histological analysis, and in vivo/in vitro functional assays. Rotenone (Rot)-pretreated UMSC-apoVs were used to explore the role of mitochondrial transfer between skin mesenchymal stem cells (SMSCs) and UMSC-apoVs in wound healing. UMSC-apoVs significantly accelerated wound healing in 5-FU-treated mice, as demonstrated by enhanced wound closure rates and histological findings of reduced inflammatory infiltration and increased collagen deposition. UMSC-apoVs transferred mitochondria to SMSCs, enhancing viability, proliferation, and migration while reducing reactive oxygen species (ROS) production in SMSCs. Rot pretreatment inhibited the therapeutic effects of UMSC-apoVs on wound healing by inducing mitochondrial dysfunction in UMSC-apoVs. Our findings indicate that UMSC-apoVs improve 5-FU-induced impaired skin wound healing by facilitating mitochondrial transfer, suggesting a novel therapeutic strategy for alleviating chemotherapy-induced impairment in wound healing.