Mammalian β-defensins are small cationic peptides that have been implicated in mediating innate immune defenses against microbial infection. Mouse β-defensin-14 (MBD-14), based on structural and functional similarities, appears to be an ortholog of human β-defensin-3 (HBD-3). Previous studies identified signaling pathway p38 mitogen-activated protein kinase (MAPK) that contributed to the expression of MBD-14 in mouse osteoblasts upon contacted with methicillin-resistance Staphylococcus aureus (MRSA) supernatant, which provided a theoretical basis as a promising therapeutic target in the treatment of intramedullary infection with MRSA in vivo. In this study, the medullary cavities of tibiae were contaminated with MRSA 10(3) colony forming units and different doses of p38 MAPK agonists anisomycin were followed as group III or IV in 30 mice. Fifteen animals that received phosphate- buffered saline served as group II and 15 mice were not contaminated with MRSA and received phosphate-buffered saline served as controls (group I). Follow-up was 7 days. In day 1, day 4 and day 7 postoperatively, infection was evaluated by blood routine, microbiological and histological analyses after sacrifice. All animals of group II developed microbiological and histological signs of infection. Histological signs of infection, white blood counts and cultures of group III and IV showed significantly reduced bacterial growth compared to cultures of group II. Simultaneously, different doses of anisomycin significantly induced the expression of osteoblast-associated genes, including alkaline phosphatase, osteocalcin and collagen type I. In addition, the expression of HBD-3 in human interfacial membranes around infected periprosthetic joint by staphylococcus contaminated was evaluated, and the expression pattern changed with significant induction of HBD-3 in infected periprosthetic joint compared with aseptic loosening under inflammatory conditions. Our primary study indicated that the potential antibacterial role of increased MBD-14 in the osteomyelitis mouse model.