Objective: Determination of the serum heat shock protein 27 (Hsp27) antibody titers and prooxidant-antioxidant balance (PAB) in patients with thalassemia as markers of cell and oxidative stress, respectively. Methods: Serum PAB and anti-Hsp27 antibody titers were measured in 140 patients with thalassemia major and 140 sex- and age-matched healthy volunteers. Results: A significantly higher serum PAB value was observed in patients in comparison to controls. In the patient group, anti-Hsp27 antibody titers were significantly higher than for the control group (p < 0.001). We found a weak negative correlation between anti-Hsp27 antibody concentrations and the PAB (p = 0.03), but these values were not correlated with serum superoxide dismutase activity in the thalassemic patients. Conclusions: Increased levels of serum PAB and Hsp27 antibodies may be involved in the pathological consequences of β-thalassemia major and may contribute to the development of endothelial injury.