Abstract: Objective To investigate the value of anti-nuclear antibody ( ANA) in the diagnosis of male patients with autoimmune liver diseases and the role of liver pathology in the examination of autoimmune liver diseases. Methods A total of 84 male patients with unexplained liver diseases who visited Nanjing Second Hospital from December 2016 to June 2018 were enrolled, and among them, 36 had positive ANA and 48 had negative ANA. The two groups were compared in terms of liver pathology, laboratory examination, and diagnosis. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. The Kruskal-Wallis H test was used for comparison of continuous data with skewed distribution between multiple groups, and the Nemenyi test was used for further comparison between two groups. The chi-square test was used for comparison of categorical data between groups. Results Among the 36 male patients in the positive ANA group, 5 had autoimmune hepatitis and 1 had overlap syndrome; among the patients in the negative ANA group, 5 had autoimmune hepatitis and 5 had overlap syndrome. There was no significant difference between the two groups in the proportion of patients with a confirmed diagnosis of autoimmune liver diseases ( 16. 7% vs 20. 8%, χ2= 0. 23, P > 0. 05) . All the patients were divided into autoimmune hepatitis group with 10 patients, overlap syndrome group with 6 patients, drug-induced liver injury group with 17 patients, and other group with 51 patients, and there were significant differences in immunoglobulin G and globulin between groups ( F = 2. 977 and 6. 972, P < 0. 05) . Interface hepatitis was observed in all ten patients with autoimmune hepatitis. Four patients in the positive ANA group and three in the negative ANA group had lymphocyte-plasma cell infiltration. Rosette-like changes of hepatocytes were found in two patients each in the positive ANA group and the negative ANA group. Lymphocyte penetration was observed in two patients and multinucleated giant hepatocytes were seen in one patient. The six patients with overlap syndrome had the pathological manifestation of moderate to severe interface hepatitis or debris-like necrosis and inflammatory infiltration in the portal vein area; among these patients, two had rosette-like changes of hepatocytes, and two had interlobular bile duct injury, lymphocyte infiltration/granuloma formation around the bile duct, and secondary hepatic fibrosis. Conclusion Positive or negative ANA has little significance in the diagnosis of autoimmune liver diseases ( autoimmune hepatitis and overlap syndrome) in ANA-positive male patients with unexplained liver diseases. Liver pathological results should be incorporated into the comprehensive analysis, which may help with the diagnosis of liver diseases.