INTRODUCTION: Antinuclear antibodies (ANA) exhibit diverse specificities and are crucial biomarkers in autoimmune disease assessment. Among ANA, anti-centromere protein-F (CENP-F) antibodies have garnered interest due to their association with malignancies. This study aims to characterize the clinical and biological profiles of a large cohort of anti-CENP-F positive patients and evaluate associated diagnoses. METHODS: Over 12 years, approximately 151,000 ANA screening samples were assessed, identifying 110 anti-CENP-F positive patients. Clinical and biological characteristics, including demographics, comorbidities, and antibody titers, were retrospectively analyzed. Follow-up data were collected to ascertain associated diagnoses. RESULTS: The median age was 50.9 years, with a predominance of women (73.6%). Median CENP-F antibody titer was 1:160, with 16.4% of patients with a titer higher than 1:1280. A concomitant specific auto-immune antibody was detected in 29 (26.4%) of cases. After a median follow-up of 3.8 years, autoimmune diseases were the most common associated diagnosis (38.2%), followed by neurological diseases (15.5%), hemopathies (8.3%), and cancer (10.9%). Some patients had concurrent cancer and autoimmune diseases. Twelve patients were diagnosed with cancer, primarily lung and breast cancers. In multivariate analysis, older age and higher anti-CENP-F titers were associated with cancer diagnosis. CONCLUSION: This study provides comprehensive insights into the clinical significance of anti-CENP-F antibodies. Vigilance is warranted in older patients with elevated antibody titers, as they may indicate an increased risk of cancer.