Purpose <p>High recurrence rates, significant metastatic potential, and limited overall survival make triple-negative breast cancer (TNBC) the most challenging subtype among breast cancers. Previous studies have indicated that the downregulation of TGFB2-AS1 can enhance the stem-like properties of tumor cells, thereby promoting TNBC progression. Bioinformatics analysis has revealed the regulatory role of GATA6 in TGFB2-AS1 transcription, providing insights into the transcriptional regulation of TGFB2-AS1 by GATA6 and offering potential prognostic biomarkers and therapeutic strategies for TNBC.</p> Methods <p>Bioinformatics analysis, Western blot, and qPCR were employed to assess the expression of GATA6 and TGFB2-AS1. Immunohistochemistry (IHC) and RNA in situ hybridization (ISH) were performed on clinical samples to evaluate GATA6 and TGFB2-AS1 expression, respectively, with survival analysis based on follow-up data. Fluorescence in situ hybridization (FISH), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays were used to elucidate the regulatory mechanisms of GATA6 on TGFB2-AS1. Functional experiments, Western blotting, qPCR, and tail vein metastasis assays, were conducted to investigate the role of GATA6-regulated TGFB2-AS1 in TNBC.</p> Results <p>GATA6 binds to the TGFB2-AS1 promoter and represses its transcription, and patients with tumors exhibiting high GATA6 and low TGFB2-AS1 expression are associated with poor prognosis. Both in vivo and in vitro functional experiments confirmed that TGFB2-AS1 critically mediates the tumor-promoting effects of GATA6 in TNBC progression.</p> Conclusions <p>Our findings reveal that GATA6 drives the progression of TNBC by repressing TGFB2-AS1 transcription.</p>