Here, we investigated the pathogenesis of primary biliary cirrhosis (PBC) by using 2D-DIGE to analyze serological differences between anti-mitochondrial antibody (AMA)-positive and -negative PBC patients. The study comprised 30 patients with PBC; 20 AMA-positive and ten AMA-negative patients matched for age, sex, and pathological stage. A screening group (four AMA-positive and four AMA-negative patients) was used for 2D-DIGE. Protein spots that were differently abundant between the two groups were identified via dye intensity and MS. Nine candidate proteins were identified from these spots. Western blotting was used to verify two of the identified proteins, serum amyloid P-component (SAP) and vitronectin (VN). VN levels were significantly higher in the sera of AMA-negative PBC patients (p <0.01), whereas no significant difference was found between the two groups for SAP. To our knowledge, this is the first study to use serological comparative proteomics to explore differences between AMA-positive and -negative PBC patients. VN levels were higher in AMA-negative PBC patients, and this finding could be related to the more severe bile duct destruction observed in this group.