Purpose: Recent studies have shown that periodontal ligament stem cells (PDLSCs) play a key role in periodontal regeneration. However, the origin of these cells remains unclear. Meanwhile, bone marrow is thought to be the most common source of adult stem cells in many tissues and organs. Thus, the present investigation sought to determine whether systemically delivered bone marrow-derived mesenchymal stem cells (BM-MSCs) could participate in periodontal regeneration and differentiate into periodontal-specific cells and to explore the origin of PDLSCs. Methods: Enhanced green fluorescent protein (EGFP)-labeled BMMSCs were delivered into lethally irradiated rats by intra-bone marrow (IBM) transplantation. Four weeks after transplantation, periodontal defects with and without infection of anaerobic cultured Porphyromonas gingivalis were established. The animals were killed 1, 2, 4, or 6 weeks after periodontal defect surgery. Histomorphologic analysis, direct observation with the fluorescence microscope, and immunohistochemical staining were performed to evaluate the localization and differentiation of BM-MSCs. Results: EGFP-positive BM-MSCs could be observed as early as 1 week after surgery, and the number of EGFP-positive cells reached a maximum at 2 weeks. Meanwhile, EGFP-positive cells were observed in the newly formed bone, PDL, and cementum 4 weeks after surgery. Immunohistochemical staining verified that EGFP-positive BM-MSCs could differentiate into osteoblasts. Conclusions: These findings provide direct evidence that BM-MSCs can participate in and modulate periodontal regeneration.