Abstract: The study hypothesized that oxidative by-product like hydrogen peroxide (H2O2), would reacts with hemoglobin (Hb) and help to initiate autoimmunity in patients with type 1 diabetes. To test this hypothesis, Hb was modified by reactive oxygen species (ROS). Modification incurred in Hb was characterized by physico-chemical techniques. ROS-modified Hb was found to be highly immunogenic in rabbits as compare to native Hb. The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and ROS-modified Hb were assessed. Diabetes patients (n = 39) were examined by direct binding ELISA and their results were compared with healthy age-matched controls (n = 22). High degree of specific binding by 48% of patient`s sera towards ROS-modified Hb, in comparison to its native analogue (p<0.05) was observed. Competitive inhibition ELISA reiterates the direct binding results. Collectively, ROS caused extensive damage to Hb altering its immunogenicity, the neo-epitopes, thus generated might play a role in the induction of circulating autoantibodies in type 1 diabetes mellitus.