The discovery and initial characterization 20 years ago of antinuclear autoantibodies (ANAs) presenting a dense fine speckled (DFS) nuclear pattern with strong staining of mitotic chromosomes, detected by indirect immunofluorescence assay in HEp-2 cells (HEp-2 IIFA test), has transformed our view on ANAs. Traditionally, ANAs have been considered as reporters of abnormal immunological events associated with the onset and progression of systemic autoimmune rheumatic diseases (SARD), also called ANA-associated rheumatic diseases (AARD), as well as clinical biomarkers for the differential diagnosis of these diseases. However, based on our current knowledge, it is not apparent that autoantibodies presenting the DFS IIF pattern fall into these categories. These antibodies invariably target a chromatin-associated protein designated as dense fine speckled protein of 70 kD (DFS70), also known as lens epithelium-derived growth factor protein of 75 kD (LEDGF/p75) and PC4 and SFRS1 Interacting protein 1 (PSIP1). This multi-functional protein, hereafter referred to as DFS70/LEDGF, plays important roles in the formation of transcription complexes in active chromatin, transcriptional activation of specific genes, regulation of mRNA splicing, DNA repair, and cellular survival against stress. Due to its multiple functions, it has emerged as a key protein contributing to several human pathologies, including acquired immunodeficiency syndrome (AIDS), leukemia, cancer, ocular diseases, and Rett syndrome. Unlike other ANAs, “monospecific” anti-DFS70/LEDGF autoantibodies (only detectable ANA in serum) are not associated with SARD and have been detected in healthy individuals and some patients with non-SARD inflammatory conditions. These observations have led to the hypotheses that these antibodies could be considered as negative biomarkers of SARD and might even play a protective or beneficial role. In spite of 20 years of research on this autoantibody-autoantigen system, its biological and clinical significance still remains enigmatic. Here we review the current state of knowledge of this system, focusing on the lessons learned and posing emerging questions that await further scrutiny as we continue our quest to unravel its significance and potential clinical and therapeutic utility.