Objective To explore the effect of human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes on fracture healing and its related mechanisms. Methods Twelve male SD rats with tibial fracture were randomly divided into 3 groups with 4 rats in each group. The rats in control group were treated with phosphate buffer solution by intra-bone marrow injection on 7 day after fracture operation, the rats in hucMSC-exosome group were treated with hucMSC-derived exosomes, and the rats in hucMSC-supernatant group were treated with exosome-free hucMSC supernatant. After treatment for 3 weeks, the healing of fracture gap was evaluated by micro-CT examination and tissue section H-E staining, and the expression levels of osteogenesis-related genes osteocalcin (OCN), osteopontin (OPN), alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx-2) were detected by qPCR. Results Micro-CT examination showed that fracture was not been joined in the control group and the hucMSC-supernatant group, with clear fracture line and discontinuous wall of cortical bone, while fracture was anastomosed in the hucMSC-exosome group, with continuous wall of cortical bone and disappeared fracture line. Tissue section H-E staining showed no fibrous callus or structure of new bone trabeculae in the control group and the hucMSC-supernatant group, while complete fibrous callus was formed and structure of new bone trabeculae was in order in the hucMSC-exosome group. The expression levels of OCN, OPN, ALP, and Runx-2 in the hucMSC-exosome group were significantly higher than those in the control group and the hucMSC-supernatant group (all P<0.05). Conclusion HucMSC-derived exosomes can promote fracture healing of rats, which may be related to the upregulated expressions of OCN, OPN, ALP, and Runx-2.