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Objective: We analyzed the localization and expression of Cluster of differentiation 40 ligand (CD40L) in murine periodontal tissue applied with the orthodontic force to determine the CD40L‐expressing cells under mechanical stress. Furthermore, we investigated whether CD40‐CD40L interaction played an important role in transducing mechanical stress between periodontal ligament (PDL) cells and cementoblasts and remodeling the periodontal tissue for its homeostasis. Background: PDL is a complex tissue that contains heterogeneous cell populations and is constantly exposed to mechanical stress, such as occlusal force. CD40 is expressed on PDL cells and upregulated under mechanical stress. However, whether its ligand, CD40L, is upregulated in periodontal tissue in response to mechanical stress, and which functions the CD40‐CD40L interaction induces by converting the force to biological functions between the cement‐PDL complex, are not fully understood. Methods: The orthodontic treatment was applied to the first molars at the left side of the upper maxillae of mice using a nickel‐titanium closed‐coil spring. Immunohistochemistry was performed to analyze the localization of CD40L in the periodontal tissue under the orthodontic force. Human cementoblasts (HCEM) and human PDL cells were stretched in vitro and analyzed CD40L and CD40 protein expression using flow cytometry. A GFP‐expressing CD40L plasmid vector was transfected into HCEM (CD40L‐HCEM). CD40L‐HCEM was co‐cultured with human PDL cells with higher alkaline phosphatase (ALP) activity (hPDS) or lower ALP (hPDF). After co‐culturing, cell viability and proliferation were analyzed by propidium iodide (PI) staining and bromodeoxyuridine (BrdU) assay. Furthermore, the mRNA expression of cytodifferentiation‐ and extracellular matrix (ECM)‐related genes was analyzed by real‐time PCR. Results: Immunohistochemistry demonstrated that CD40L was induced on the cells present at the cementum surface in periodontal tissue at the tension side under the orthodontic treatment in mice. The flow cytometry showed that the in vitro‐stretching force upregulated CD40L protein expression on HCEM and CD40 protein expression on human PDL cells. Co‐culturing CD40L‐HCEM with hPDF enhanced cell viability and proliferation but did not alter the gene expression related to cytodifferentiation and ECM. In contrast, co‐culturing CD40L‐HCEM with hPDS upregulated cytodifferentiation‐ and ECM‐related genes but did not affect cell viability and proliferation. Conclusion: We revealed that in response to a stretching force, CD40L expression was induced on cementoblasts. CD40L on cementoblasts may interact with CD40 on heterogeneous PDL cells at the necessary time and location, inducing cell viability, proliferation, and cytodifferentiation, maintaining periodontal tissue remodeling and homeostasis. [ABSTRACT FROM AUTHOR]