Background <p>Dental follicle stem cell (DFSC) therapy is an excellent option for reducing alveolar bone loss. Preconditioning DFSCs increases their resistance to the disease microenvironment and increases their efficacy. Photobiomodulation therapy (PBMT) is an effective and convenient way to precondition DFSCs. In this study, we evaluated the effects of PBMT-pretreated DFSCs on alveolar bone regeneration and investigated the underlying mechanism.</p> Methods <p>After DFSCs were isolated and characterized, various biological properties of DFSCs following treatment with PBMT were evaluated in viability tests, wound healing assays, alizarin red staining, alkaline phosphatase staining, real-time quantitative PCR, mitochondrial membrane potential assays, and ATP and reactive oxygen species (ROS) measurements. Twenty-four 7-week-old SD rats were used to establish a periodontal defect model and were then divided into 3 groups: the control group, the DFSC group, and the PBMT + DFSC group. Microcomputed tomography and histological examination were conducted at two and four weeks.</p> Results <p>In the in vitro study, the PBMT group showed better osteogenic capability than the control group. The gene expression levels of COL-1α, OCN, and RUNX2 in the PBMT group increased. Moreover, ROS and ATP levels and the mitochondrial membrane potential were greater in the PBMT group than in the other groups. In the in vivo study, the bone volume per total volume (BV/TV) in the PBMT + DFSC group was greater than those in the control and PBMT groups at four weeks. There was more new bone tissue, more trabeculae and higher expression of OCN and COL-I in the slices of the PBMT + DFSC group than in those of the other groups.</p> Conclusions <p>PBMT facilitated bone regeneration in periodontal defect models by enhancing the osteogenic and mitochondrial functions of DFSCs.</p>