The use of biological repair in infectious bone defects has been a major challenge for clinicians. With potential for bone regeneration, stem cell therapy could be an effective biological restoration measure for infection-caused bone destruction. In this study, we propose a new stem cell therapy strategy for infectious bone defect repair through systemic transplantation of human dental pulp stem cells (hDPSCs). Hypoxic preconditioning (HP) is thought to be able to enhance duration of survival and therapeutic potency of engrafted stem cells; therefore, we examined the role of HP on hDPSC therapeutic efficacy. Our results show that HP significantly enhanced hDPSC survival rate and osteogenic differentiation. hDPSCs were all CXCR4 positive under hypoxic pretreatment and their migration in response to SDF-1 was increased in vitro. hDPSC migration increase can be abolished after application of CXCR4 antagonist, AMD3100. In a mouse apical periodontitis bone destruction model, after transplantation of hypoxic preconditioned hDPSCs through intravenous injection, upregulated hDPSC recruitment and recovery of alveolar bone mass were observed in infected periapical tissue, and osteogenesis and bone mineralization were enhanced. Significantly, in periapical lesions, we found increased SDF-1 production and CXCR4/CFSE+ colabeled cells. Together, our results suggested that hypoxic preconditioned hDPSCs are capable of repairing infectious bone defects through the SDF-1/CXCR4 axis. Our investigation provides a novel infection-caused bone loss therapeutic strategy using hDPSC transplantation, and HP is an effective way of improving hDPSC survival rate, recruitment, and osteogenesis.