Background <p>Periodontitis, the leading cause of tooth loss worldwide, is closely linked to the compromised regenerative capacity of human periodontal ligament stem cells (hPDLSCs). Stem cell-based tissue engineering is a promising treatment for periodontitis. Sustaining the osteogenic potential of hPDLSCs against adverse conditions following transplantation is critical for successful periodontal tissue engineering. Recent research increasingly underscores ferroptosis as a crucial target for periodontitis treatment, whereas reactive oxygen species (ROS) contribute to ferroptosis initiation and progression. Klotho, an anti-aging protein, has been shown to protect hPDLSC osteogenic function under oxidative stress in our previous study. However, whether Klotho can provide protection against ferroptosis and maintain osteogenic function of hPDLSCs in an inflammatory environment remains elusive.</p> Methods <p>Ferroptosis level and the expression of Klotho in hPDLSCs under normal and inflammatory conditions were compared via single-cell RNA sequencing and validation experiments. Stable Klotho-overexpressing hPDLSCs (hPDLSCs-ov-KL) cell line was established and the impact of Klotho on ferroptosis was assayed. Subsequently, the effect of Klotho overexpression on hPDLSC osteogenesis was evaluated under in vitro inflammatory environment and in vivo periodontitis model of C57BL/6 mice. Additionally, the underlying molecular mechanism of Klotho effect on hPDLSCs under the inflammatory environment was investigated.</p> Results <p>Ferroptosis was activated and the expression of Klotho was reduced in hPDLSCs under LPS-stimulated inflammatory environment, consistent with the results in hPDLSCs of periodontitis via single-cell RNA sequencing. Further experiments confirmed Klotho overexpression effectively suppressed ferroptosis in hPDLSCs and markedly preserved the hPDLSC osteogenic capacity under in vitro inflammatory environment. In vivo, injection of hPDLSCs-ov-KL could effectively promote periodontal tissue repair in the mouse model of periodontitis. From the perspective of molecular mechanism, Klotho notably inhibited NOX4 expression in hPDLSCs under the inflammatory environment and NOX4 overexpression in hPDLSCs-ov-KL significantly increased intracellular ferroptosis, leading to compromised Klotho protective effect.</p> Conclusion <p>Our study highlighted the significant protective effect of Klotho on counteracting hPDLSC ferroptosis via the inhibition of NOX4 expression, therefore restoring the impaired osteogenic function of hPDLSCs in both in vitro inflammatory environment and in vivo periodontitis model, which might provide a promising strategy for periodontal tissue regeneration engineering.</p> Graphical Abstract <p></p>