Anti-ribonucleoprotein (anti-RNP) antibodies are one of the representative autoantibodies detectable in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Generally, posttranslational modifications (PTMs) on autoantigens are proposed to be involved in the production of autoantibodies. In this study, we tried to detect the alteration in PTMs on a U1 small nuclear RNP 68k subunit (U1-68k), a major antigen of anti-RNP antibodies. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with MCTD, SLE, and rheumatoid arthritis (RA), and from healthy donors. U1-68ks in the PBMCs were detected by 2D Western blot (WB), where extracted nuclear proteins were separated by 2DE, followed by the detection of U1-68k using WB. More than 20 PTM isoforms were detected with different molecular weights of 65.0 , 66.5, and 68.0kDa, and different pIs between 6.0 and 8.5. Importantly, the relative intensity of the spot with 66.5 kDa and pI 7.5 was significantly increased in the MCTD and SLE groups compared to the RA and healthy groups. Further, this U1-68k isoform, in particular, in its RS domain, was found to have significantly decreased phosphorylation compared to the other isoforms. The PTM alternation may be one of the steps to generate the anti-RNP antibodies.