Thiamine deficiency causes dysfunction in the heart (wet beriberi) and nervous system and is considered a health problem. This study was designed to investigate the production of reactive oxygen species (ROS), the activity of antioxidant enzymes and their protein expression in rat heart tissue during thiamine deficiency. We also aimed to determine the occurrence of apoptosis. Male Wistar rats weighing 200-250g were kept at 25°C with light/dark cycle of 12 hours and fed a diet deficient in thiamine for 35 days. After, the control (CT) and thiamine-deficient (TD) rats were killed and the hearts were removed for carrying out the proposed analyses. This study was approved by the Institutional Ethics Committee No. 042/2006. Superoxide-dismutase (SOD) activity was performed according to Dieterich et al. (2004) with minor modifications. SOD activity was calculated as units per milligram of protein and one unit of enzyme was considered as being the amount that caused inhibition of pyrogallol autoxidation by 50%. To evaluate Glutathione-peroxidase (GPx) activity the tissue samples were homogenized in ice-cold Tris-HCl buffer and centrifuged at 4°C for 30 min. Results were expressed in nmol NADPH/min/mL. Catalase (CAT) activity was measured in the supernatants as described by Nelson & Kiesov (1971) and its activity expressed by ΔE/min/mg protein. The expression of enzymes was also analyzed by Western blot (n=3). Measurement of intracellular superoxide and H2O2 levels were performed in isolated cardiomyocytes previously loaded with 10μM DHE and with 0.1μM of 2,7-dichlorodihydrofluorescein-diacetate. To measure apoptosis, cardiac samples were fixed in buffered 10% formalin, processed routinely for histopathology, sectioned at a 5 μm and stained. Another set of slides were submitted either to TUNEL or Caspase-3 assays to detect apoptosis. Sample comparisons were performed using Student’s t test or one way ANOVA followed by post-hoc analysis. p< 0.05 was used as a measure of statistical significance. The results showed an increased production of ROS in TD rats. The activity of SOD was not changed (CT: 0.16 ± 0.003 U.mg protein-1 (n = 5) vs TD: 0.15 ± 0.006 U.mg protein-1 (n = 6)), but its expression decreased. Protein expression and activity (CT:1.14 ± 0.192 ΔE.min-1.mg-1(n=6) vs TD: 0.402 ± 0.096 ΔE.min-1.mg-1(n=6)) for CAT increased 2.1 fold in TD rats compared to control animals. We found that GPx activity decreased (CT: 435.3 ± 28.6 nmol NADPH.min-1.ml-1(n=6) vs DT: 199.4 ± 30.2 nmol NADPH.min-1.ml-1(n=6)) as well as protein expression in TD rats. Apoptosis was less intense in the control group than in the TD group. Taken together, we can conclude that in thiamine deficiency oxidative stress is increased by altering the activity and expression of antioxidant enzymes and therefore leads to apoptosis which could explain the cardiac dysfunction previously observed.