Calprotectin, a heterodimer of S100A8 and S100A9 molecules, is associated with inflammatory diseases such as inflammatory bowel disease. We have reported that calprotectin levels in gingival crevicular fluids of periodontitis patients are significantly higher than in healthy subjects. However, the functions of calprotectin in pathophysiology of periodontitis are still unknown. The aim of this study is to investigate the effects of calprotectin on the productivity of inflammatory cytokines in human gingival fibroblasts (HGFs). The HGFs cell line CRL‐2014® (ATCC) were cultured, and total RNAs were collected to examine the expression of TLR2/4 and RAGE mRNA using RT‐PCR. After the cells were treated with S100A8, S100A9, and calprotectin, supernatants were collected and the levels of IL‐6 and MCP‐1 were measured using ELISA methods. To examine the intracellular signals involved in calprotectin‐induced cytokine production, several chemical inhibitors were used. Furthermore, after the siRNA‐mediated TLR4 down‐regulated cells were treated with S100A8, S100A9, and calprotectin, the levels of IL‐6 and MCP‐1 were also measured. HGFs showed greater expression of TLR4 mRNA, but not TLR2 and RAGE mRNA compared with human oral epithelial cells. Calprotectin increased significantly the production of MCP‐1 and IL‐6 in HGFs, and the cytokine productions were significantly suppressed in the cells treated with MAPKs, NF‐κB, and TLR4 inhibitors. Furthermore, calprotectin‐mediated MCP‐1 and IL‐6 production were significantly suppressed in TLR4 down‐regulated cells. Taken together, calprotectin induces IL‐6 and MCP‐1 production in HGFs via TLR4 signaling that involves MAPK and NF‐κB, resulting in the progression of periodontitis. J. Cell. Physiol. 232: 1862–1871, 2017. © 2016 Wiley Periodicals, Inc.