•STIL is upregulated in breast cancer, and promoted the proliferation, migration and invasion of triple negative breast cancer.•Our findings, for the first time, provide novel insights into the regulatory role of STIL in the FA pathway within breast cancer.•Our findings identify STIL as a critical promoter of breast cancer progression by interacting with KLF16 to regulate FANCD2. STIL is an important cell cycle-regulating protein specifically recruited to the mitotic centrosome to promote the replication of centrioles in dividing cells. However, the potential role of STIL in the regulation of the biological functions of triple-negative breast cancer remains still unclear. We screened for differentially expressed STIL in the Cancer Genome Atlas database. The expression of STIL protein in 10 pairs of breast cancer tissues and adjacent normal tissues was further assessed by western blotting. Functionally, the knockdown and overexpression of STIL have been used to explore the effects of STIL on breast cancer cell proliferation, migration, and invasion. Mechanistically, RNA-seq, dual-luciferase reporter assay, chromatin immunoprecipitation assay, mass spectrometry, immunoprecipitation assay, and DNA pull-down assay were performed. Breast cancer tissues and cells have higher STIL expression than normal tissues and cells. STIL knockdown impairs breast cancer cell growth, migration, and invasion, whereas STIL overexpression accelerates these processes. STIL promotes breast cancer progression by regulating FANCD2 expression, and exploration of its molecular mechanism demonstrated that STIL interacts with KLF16 to regulate the expression of FANCD2. Collectively, our findings identified STIL as a critical promoter of breast cancer progression that interacts with KLF16 to regulate Fanconi anemia pathway protein FANCD2. In summary, STIL is a potential novel biomarker and therapeutic target for breast cancer. STIL is significant overexpression in triple-negative breast cancer. And STIL interacts with KLF16 protein, thereby facilitating the binding of KLF16 to the FANCD2 promoter and promoting transcriptional activation of FANCD2 Display omitted