The role of aberrant protein modifications in cancer and its diagnosis have emerged as a promising research field. Nonenzymatic glyco-oxidation of proteins under oxidative stress has been associated with carcinogenesis through advanced glycation end products (AGE)–receptors for advanced glycation end products (RAGE) axis. Modified proteins that are immunogenic and stimulate cellular and humoral immune responses are being studied to develop early detection markers of cancer. This study has probed the structural alternations; leading to the formation of adducts and aggregates, in histone H2A upon in vitro modification by methylglyoxal (MG). The immunogenicity of modified histone H2A and its binding with cancer autoantibodies was also assessed. MG induced lysine side chain modifications, blocking of free amino groups and the formation of condensed cross structures in histone H2A; and its effect was inhibited by carbonyl scavengers. It led to the adduct formation and generation of N-epsilon-(carboxyethyl)lysine (CEL) and its decomposition forms as revealed by Matrix-assisted laser desorption ionization–mass spectrometry, high-performance liquid chromatography and LC–MS. MG-H2A showed amorphous aggregate formation under electron microscopy and altered binding with DNA in circular dichroism studies. The modified histone elicited high titer immunogen-specific antibodies in rabbits when compared with the native, thus pointing toward the generation of neo-epitopes in MG-H2A. The autoantibodies derived from cancer patients exhibited enhanced binding with MG-H2A as compared with the native histone in enzyme-linked immunosorbent assay and gel retardation assay. This reflects sharing of epitopes on MG-H2A and histones in cancer patients. The neo-epitopes on H2A may be responsible for induction and elevated levels of antibodies in cancer patients. Thus, MG-H2A may be considered as potential antigenic candidate for auto-immune response in cancer.