Breast cancer is the second leading cause of death in women worldwide. Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with poor prognosis, high recurrence rate, and lack of effective treatment therapies. Super-enhancers have been shown to play a key role in upregulating oncogenes in various cancers. However, the subtype-specific function of super-enhancers in breast cancer has not been studied. Understanding the regulation of genes by super-enhancers will provide a deeper insight into breast cancer biology, clinical diagnosis, and anti-cancer therapy. We have analyzed H3K27ac ChIP-seq data from GEO and ENCODE and uncovered a TNBC-specific super-enhancer proximal to ANLN. Playing a key role in cytogenesis, ANLN is overexpressed in many cancers. The role of ANLN in TNBC and the downstream pathways regulated by ANLN during tumorigeneses, however, remain elusive. ANLN is upregulated in a panel of TNBC lines as well as in clinical TNBC samples. In addition, upregulation of ANLN is a predictor of poor survival in breast cancer patients. To assess the regulatory effect of super-enhancer on ANLN expression, CRISPER/Cas9 genomic editing technique was utilized to delete the peak of super-enhancer proximal to ANLN gene. The deletion of super-enhancer reduced the protein expression level of ANLN accompanied by decreased spheroid and colonogenic growth in TNBC cell lines, indicating the functional importance of super-enhancer in upregulating oncogenic protein expression and tumorigenesis. Using CRISPER/Cas9-mediated inducible knockout of ANLN, we showed that ANLN depletion inhibited TNBC spheroid growth in 3D cultures. ANLN depletion also significantly reduced the progeny producing capability of cells as assessed by colonogenic assay. By performing mammosphere assay, we further demonstrated the critical function of ANLN in tumor-initiating capability in vitro. This prompted us to explore the unknown mechanism by which ANLN regulates breast cancer stemness. Using RT-qPCR, a total of 49 cancer stem cell markers were screened to uncover the potential target genes of ANLN. Expression levels of five genes were reduced in ANLN-knockout TNBC cells consistently. These genes include Twist1, Bmp2, Notch1 and Notch3 and FOXK1. Rescue experiments revealed that Twist1, Bmp2, Notch1 and Notch3 were able to restore mammosphere formation ability in ANLN-depleted TNBC cells. This study elucidated the regulation of ANLN expression by TNBC-specific super-enhancer and identified the importance of Twist1 and Bmp2 as effectors of ANLN in promoting cancer stem cell properties, which will provide insight for the development of targeted therapy for TNBC patients.