Background The gingival epithelium protects periodontal tissues and the alveolar bone by maintaining a steady state of regulated inflammatory surveillance, also known as healthy homeostasis. Accordingly, the repertoire of receptors present within the gingival epithelium showcases its ability to recognize microbial colonization and contribute to bacterial sensing. Macrophage migration inhibitory factor (MIF) is one of many cytokines that are expressed in this protective state and is involved in neutrophil regulation. However, its role in the maintenance of healthy gingival tissue has not been described. Methods Gingival tissues from wild‐type (WT) and Mif knock‐out (KO) mice were stained for neutrophils and three key neutrophil chemoattractants: MIF, Gro‐α/CXCL1, and Gro‐β/CXCL2 in the junctional epithelium (JE). In addition, gene silencing studies were performed using gingival epithelial cells (GECs) to examine the role of MIF on transcription of key bacterial recognition receptors Toll‐like receptors (TLR)‐1, ‐2, ‐4, ‐6, ‐9 and interleukin‐1 receptors (IL‐1R1 and IL‐1R2) in response to oral bacterial stimulation. Results WT murine gingival tissues demonstrated high expression of MIF in the JE. In Mif KO mice, despite the significant reduction of Gro‐α/CXCL1 and Gro‐β/CXCL2, there was a slight increase in neutrophils. Gene silencing experiments showed that MIF down‐regulated the mRNA expression of TLR4, IL‐1R1, and IL‐1R2 in GEC, in addition to decreasing secreted IL‐8/CXCL8 in response to bacteria. Conclusions MIF regulates the expression of TLR4, IL‐1Rs, and IL‐8/CXCL8, components that are all involved in maintaining oral health. Our data demonstrate that MIF is a significant contributor to the maintenance of healthy oral homeostasis.