Chronic periodontitis (CP) is an infection that affects the teeth supporting structure. Macrophage migration inhibitory factor (MIF) is an important effector cytokine of the innate immune system. Due to its functional characteristics, MIF may be involved in the immunopathology of CP. The aim of the present study was to evaluate MIF levels in gingival crevicular fluid (GCF), saliva, and serum of CP patients. A cross-sectional study was conducted on 60 subjects divided into two groups: subjects with CP (n= 30) and periodontally healthy subjects without CP (n=30). MIF was quantified in GCF, saliva, and serum of all participants by enzyme-linked immunosorbent assay. MIF concentrations were higher in GCF, saliva, and serum in the group with CP compared with the group without CP and a higher MIF concentration was observed in GCF (p=0.001) and saliva (p=0.009) in the group with CP. MIF intragroup comparisons between fluids demonstrated significant high levels of MIF in saliva compared with GCF and serum in both study groups (p<0.05). A positive correlation was found between clinical signs and MIF concentration in GCF (p<0.05). There is an association between the MIF and the clinical signs of the disease. Therefore, MIF could have an important role in the pathology and progression of CP.