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Metabolic syndrome (MetS) is characterized by clustering of clinical, physiological, biochemical, and metabolic factors that are associated with an increased risk of cardiovascular disease (CVD) and type 2 diabetes mellitus. Immune response to heat shock protein 27 (Hsp27) has been suggested to be implicated in atherogenesis. We aimed to investigate the association between serum anti-Hsp27 antibody concentrations and type 2 diabetes in patients with MetS. This was a cross-sectional observational study on groups of MetS and healthy subjects. The population sample was derived from MASHAD STUDY, a national cross-sectional study conducted by the Ministry of Health and Medical Education in 2004. Pregnant and breastfeeding women and patients who had cardiovascular disease, myocardial infarction, stroke, or systemic disease were excluded from the 9600 subjects of the MASHAD STUDY population. A total of 933 subjects including 477 women and 456 men were classified as having MetS, diabetes mellitus, or neither. Data including age, gender, and smoking habit collected using a questionnaire. MetS was diagnosed based on the International Diabetes Federation (IDF) definition. The serum anti-HSP27 antibody titers were measured by ELISA. There was no difference in serum anti-HSP27 concentrations between subjects with and without MetS, or diabetes mellitus, nor was there a significant difference in anti-HSP27 levels between men and women. There was no significant difference in anti-HSP27 antibody between diabetic MetS patients, normal population, non-diabetic MetS, and diabetic non-MetS patients (p value >0.05). Serum anti-Hsp27 antibody concentrations did not differ between individuals with or without MetS or diabetes mellitus.