BackgroundHuman periodontal ligament stem cells (hPDLSCs) have a superior ability to promote the formation of new bones and achieve tissue regeneration. However, mesenchymal stem cells (MSCs) are placed in harsh environments after transplantation, and the hostile microenvironment reduces their stemness and hinders their therapeutic effects. Klotho is an antiaging protein that participates in the regulation of stress resistance. In our previous study, we demonstrated the protective ability of Klotho in hPDLSCs.MethodsA cranial bone defect model of rats was constructed, and the hPDLSCs with or without Klotho pretreatment were transplanted into the defects. Histochemical staining and micro-computed tomography were used to detect cell survival, osteogenesis, and immunoregulatory effects of hPDLSCs after transplantation. The in vitro capacity of hPDLSCs was measured by a macrophage polarization test and the inflammatory level of macrophages. Furthermore, we explored autophagy activity in hPDLSCs, which may be affected by Klotho to regulate cell homeostasis.ResultsPretreatment with the recombinant human Klotho protein improved cell survival after hPDLSC transplantation and enhanced their ability to promote bone regeneration. Furthermore, Klotho pretreatment can promote stem cell immunomodulatory effects in macrophages and modulate cell autophagy activity, in vivo and in vitro.ConclusionThese findings suggest that the Klotho protein protects hPDLSCs from stress after transplantation to maintain stem cell function via enhancing the immunomodulatory ability of hPDLSCs and inhibiting cell autophagy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13018-023-03849-8.