Dental follicle stem cells (DFSCs) have been verified to promote periodontal regeneration in an inflammatory microenvironment. When coping with inflammatory stimulation, DFSCs highly express periostin, a bioactive molecule closely related to periodontal homeostasis. It is worth exploring whether and how periostin plays a role in the promotion of periodontal regeneration by DFSCs. By tracking the fate of DFSCs, it was found that DFSCs significantly contributed to periodontal regeneration in rat periodontal defects while they had a low survival rate. They highly expressed periostin and improved the immune microenvironment in the defect area, especially via the recruitment and reprogramming of macrophages. Silencing periostin attenuated the effects of DFSCs in promoting periodontal regeneration and regulating macrophages. Recombinant human periostin (rhPeriostin) could not only directly promote macrophage reprogramming through the integrin αM/phosphorylated extracellular signal-regulated kinase (p-Erk)/Erk signaling pathway, but it also exhibited the potential to promote periodontal regeneration in rats when loaded in a collagen matrix. These results indicated that periostin is actively involved in the process by which DFSCs promote periodontal regeneration through the regulation of macrophages and is a promising molecular agent to promote periodontal regeneration. This study provides new insight into the mechanism by which DFSCs promote periodontal regeneration and suggests a new approach for periodontal regeneration therapy.