Objective: Calprotectin, the heterdimer of S100A8 and S100A9, is the major cytoplasmic protein of neutrophils, which is also expressed or induced in gingival epithelial cells, activated mononuclear macrophages and vascular endothelial cells. Calprotectin is intimately associated with the initiation and progression of periodontitis, but the in vivo expression patterns of calprotectin in healthy and inflamed periodontal tissue are not fully understood. To observe the expression, distribution and cellular localization of calprotectin in the samples of healthy periodontal tissues and experimental periodontitis tissues of Beagles and to explore their relationship with periodontal inflammation and possible effect. Methods: Experimental periodontitis model was established by ligation around the mandibular second molar of the Beagle dogs, while the contralateral teeth were healthy controls. Induction duration was 12 weeks, before the dogs were executed. Tissue specimens were demineralized and serial sections were made conventionally. The in vivo expression of calprotectin in the healthy and inflamed periodontal tissues were examined by immunohistochemistry. The in vitro expression of calprotectin in human primary gingival fibroblasts (GFs) and periodontal ligament (PDL) cells were detected by immunocytochemistry. Results: Immunohistochemistry analysis indicated that calprotectin was expressed in gingival epithelial cells and infiltrated neutrophils in the healthy periodontium within the gingival epithelium, S100A8/A9 was most strongly expressed in the junctional epithelium, followed by surface epithelium, and least expressed in the sulcular epithelium. The S100A8/A9 expression levels were sharply defined at the junction between the junctional epithelium and the sulcular epithelium. In periodontal inflammatory lesions, the expression level of calprotectin in sulcular epithelium and junctional epithelium was up-regulated than that in the healthy gingival epithelium. Calprotectin was inducibly expressed in fibroblast-like cells in gingival connective tissue and periodontal ligament tissue, microvascular endothelial cells (ECs) and bone marrow fibroblasts under inflammatory conditions. Additionally, the expression of calprotectin in primary human GFs and PDL cells was confirmed by immunnocytochemistry staining. Conclusion: Constitutively expressed in neutrophils and gingival epithelial cells, and calprotectin might maintain the homeostasis and integrity of periodontium. Inflammation-induced expression of calprotectin in GFs, PDL cells, microvascular ECs and bone marrow fibroblasts might process anti-microbial function and promote leukocytes transmigration to defend the host against the microorganisms.