INTRODUCTION: Thimerosal (TH) is a mercury-based organic compound used as a preservative in many vaccines. When, in the human body, it is metabolized or degraded to ethylmercury and thiosalicylate. The ethylmercury has a high affinity to thiol functions of proteins and can, therefore, bind to protein matrices like human hemoglobin because of its several free thiol functions [1]. Also, this structure can interact with human erythrocytes (Ery) affecting the O2 uptake by hemoglobin. In this context, this work evaluated the interaction of thimerosal with human hemoglobin (HbTH) through monitoring its ability to reduce the binding capacity of Hb toward O2. In these measures, Ery, from adult humans, have been exposed for two different concentration of TH (1.25 and 2.50 μM) with an incubation time of 3 min (or 5 min) for two temperatures 28 and 37°C. MATERIAL AND METHODS: Ery were used to evaluate O2 uptake, considering the following variables: TM concentration (1.25 and 2.50 μM), incubation time (3 or 5 min) and temperature (28 and 37°C), we also tested HgCl2 as a positive control. Sulfidryl content and Raman spectrum changes in the presence of TM were also evaluated. RESULTS AND DISCUSSION: In assays the time of 5 min showed the most pronounced responses in Ery, as the reduction of O2 uptake at 28°C, that was 38% (1.25 μM) and 51% (2.50 μM). At 37°C it reached 51% (1.25 μM) and 61% (2.50 μM). Mercury chloride, used as a control, showed at least 62% of reduction in O2 uptake. The -SH assays quantified the preservation of these groups up to 59% at Ery in the presence of NEM (reducing agent). In the Raman spectrum we verified differences in regions that are related to Fe-O bond and distortion of the hemoglobin porphyrinic ring. CONCLUSION: Based on our results, it is suggested that TM binds to hemoglobin causing conformational changes and these leads to a deleterious effect on its primary O2 binding function.