AbstractStem cell injection is good for periodontal regeneration due to the capacity of stem cells to differentiate toward osteogenic direction and to regulate the production of pro‐ and anti‐inflammatory cytokines. However, injected cells are difficult to track in vivo. And there is microbiota in oral cavity, the dysbiosis of which leads to the damage and loss of periodontal tissue. Here, we demonstrated an enhanced periodontal repair was due to an altered oral microbiota. Periodontal defects were surgically prepared in rats, and periodontal ligament stem cells (PDLSCs) labeled by superparamagnetic iron oxide (SPIO) nanoparticles (PC‐SPIO) were injected, with PDLSCs and saline treatments as controls. Detected by magnetic resonance imaging (MRI) and histological staining, PC‐SPIO was major at limited areas in regenerated periodontal tissues. PC‐SPIO‐treated rats achieved better periodontal regeneration than the other two groups. Concurrently, the oral microbiota of PC‐SPIO‐treated rats was changed, presenting SPIO‐Lac as a biomarker. SPIO‐Lac assisted periodontal repair in vivo, inhibited the inflammation of macrophages induced by lipopolysaccharide (LPS) and antibacterial in vitro. Therefore, our study proved that SPIO‐labeled cells can be tracked in periodontal defect and highlighted a potential positive role of an oral microbiota in periodontal regeneration, suggesting the possibility of periodontal repair promotion by manipulating oral microbiota.